Pathogen vaccines and methods of producing and using the same

ABSTRACT

The present invention provides vaccine compositions and methods of producing such compositions. Other embodiments of the invention include methods of treating a pathogen infection, methods of vaccinating a subject against a pathogen infection, and methods for treating an antibiotic-resistance bacterial infection in a subject in need thereof. In further embodiments, the invention includes methods of decreasing the level of a pathogen in a subject having a pathogen infection, methods of increasing the surviving rate of a subject having a pathogen infection, methods of reducing the level of pain associated with a pathogen infection, and methods of reducing the level of distress associated with a pathogen infection in a subject in need thereof. Novel scaffold compositions and opsonin-bound or lectin-bound pathogen compositions, and uses thereof, are also provided herein.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/343,448, filed on May 31, 2016, and U.S. ProvisionalApplication No. 62/295,711, filed on Feb. 16, 2016. The entire contentsof each of the foregoing applications are incorporated herein byreference.

GOVERNMENT SUPPORT

The invention was made with government support under DARPAN66001-11-1-4180. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 28, 2018, isnamed 117823_12203_SeqList.TXT and is 1,681 bytes in size.

BACKGROUND OF THE INVENTION

Infectious diseases are caused by a pathogenic microorganism, like avirus, bacterium, fungus, or the like which enters and propagates in aliving body. Common strategies to treat infectious diseases include theadministration of antimicrobial drugs such as antivirals or antibiotics,or the use of immunotherapy such as vaccination, to patients sufferingfrom or prone to suffering such infections.

However, in some cases, the pathogenic microorganisms can not be easilyeradicated by the use of existing antimicrobial drugs as thesemicroorganisms may acquire resistance to drugs, or the drugs may poseundesirable side effects to varying degrees to patients. As a result,known antibiotics and antivirals have not been entirely satisfactory interms of their antimicrobial activity, behavior in the body, safety, orability to suppress drug-resistant microorganisms.

Vaccines reduce the risk of infection by working with the body's naturaldefenses to help it safely develop immunity against pathogens. Althoughvaccines have been considered among the most powerful tools available topublic health, there are certain limitations associated with vaccinesfor treating or preventing infectious diseases. For example, developmentof vaccines against a pathogen infection usually requires identificationor isolation of the pathogen. In the case where the specific pathogen isunknown or isolation of a pathogen poses a great difficulty, preparationof vaccines would be of a great challenge and may take a much longertime. In addition, antigenicity of the pathogen is easily altered, andfor pathogens that have multiple strains with different surfaceantigens, inconsistency in the antigen structure between vaccine strainand the infected strain would be a significant problem. When a straindifferent from the vaccine administered causes an infectious disease,the vaccination becomes ineffective. Furthermore, pathogen leakage hasbeen observed in certain vaccines and causes undesired side effect andmassive inflammation within the subject.

As a result, infectious diseases remain a major public health threat anda leading cause of illness, disability and death across the world.Accordingly, there remains an ongoing and unmet need for the developmentof novel therapeutic strategies and vaccines to treat infectiousdiseases.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery thatpathogens or pathogen associated molecular patterns (PAMPs) isolatedusing an opsonin or lectin, e.g., an engineered lectin or fragmentthereof, can be used to generate functional vaccines for the treatmentof infectious diseases. In particular, the present inventors havesurprisingly discovered that, pathogens or pathogen associated molecularpatterns (PAMPs) isolated using an engineered lectin when combined witha bioactive agent, e.g., an adjuvant, and/or a scaffold, allow for therapid creation of high potency pathogen vaccines (FIG. 1). When used tovaccinate animals, a single dose of these vaccines resulted in asignificantly reduced pathogen titer in the vaccinated animals and asignificantly prolonged survival time after infecting the animals with alethal dose of bacteria. Indeed, as shown in Example 1, a single dose ofthe vaccine composition of the invention can protect the vaccinated micefrom a bacteria challenge over a period of 90 days. In addition, theopsonin or lectin, e.g., an engineered lectin or fragment thereof, notonly functions to isolate a pathogen for use in the vaccine compositionsand present the pathogen to immune cells to initiate an immune response,but also serves as an anchor structure to immobilize the pathogen, thuspreventing leakage of the pathogen from the vaccine composition, andpreventing any undesired side effects currently experienced withpathogen leakage.

The vaccine compositions of the present invention possess additionalimprovements over existing vaccines. For example, the vaccinecompositions of the present invention allow the rapid and directisolation of pathogens circulating in a blood sample from a patient withinfectious disease including both known and unknown pathogens, pathogenspresent within other biological fluids, or pathogens present in in vitrocultures. The claimed vaccine compositions can also be used againstpathogens that are difficult to isolate and purify. Once the pathogensare isolated from a subject, the vaccine compositions can be readilyprepared in a fast and convenient manner anywhere in the world, and canbe available for patients in a timely manner, for example, within oneday. In addition, vaccination using the claimed vaccine compositions canoccur in a more controlled, localized and safer manner, withoutcompromising the efficacy of the vaccine compositions. The improvedstability of the claimed vaccines allows them to be portable and to beused for long term storage at room temperature without the need ofrefrigeration. Furthermore, the vaccine compositions can be multivalentvaccines when more than one type of pathogens are included in thecompositions, and can also be used to vaccinate against differentspecies or strains of a given pathogen. In addition, the vaccinecompositions, if implanted, can be easily removed from the subject aftervaccination. For example, in the case where too much immune response orundesired side effects are initiated after vaccination, the implantedvaccine compositions can be readily removed from the subject. Incontrast, current existing vaccines cannot be removed once they areintroduced in the subjects. These improvements circumvent the majorlimitations of current pathogen vaccines, and would be of great interestto the public, especially during the time of an epidemic, for example,for populations in developing countries, or of great value for militaryuses, where vaccines that are readily available are highly desired.Indeed, the ability to rapidly create functional and highly stablevaccines that are not only easy for storage and handling, but may beadministered in a safer and more controlled manner and confer along-term protective effect, renders the vaccine compositions of thepresent invention significantly advantageous over existing vaccines.

Accordingly, in one aspect, the present invention provides vaccinecompositions. The vaccine compositions comprise an opsonin-bound orlectin-bound pathogen construct, and a bioagent capable of recruiting animmune cell in a subject.

In some embodiments, the bioagent is selected from the group consistingof interleukin (IL)-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10,IL-12, IL-15, IL-17, IL-18, tumor necrosis factor (TNF)-alpha,interferon (IFN)-gamma, IFN-alpha, granulocyte macrophage colonystimulating factor (GM-CSF), granulocyte colony stimulating factor(G-CSF), Fms-related tyrosine kinase ligand (FTL)-3 ligand, CCL19,CCL21, M-SCF, MIF, CD40L, CD3, ICAM, transforming growth factor(TGF)-beta, cytosine-guanosine oligonucleotide (CpG-ODN),lipopolysaccharides (LPS), Fas ligand, Trail, lymphotactin, Mannan(M-FP), APG-2, Hsp70 and Hsp90.

In some embodiments, the bioagent comprises an adjuvant. In otherembodiments, the adjuvant is selected from the group consisting ofcytosine-guanosine oligonucleotide (CpG-ODN) sequence, granulocytemacrophage colony stimulating factor (GM-CSF), ovalbumin (OVA),monophosphoryl lipid A (MPL), poly(I:C), MF59, alum, aluminum hydroxide,aluminum phosphate, calcium phosphate hydroxide, Quil A, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), FIA, montanide, adjuvant 65,lipovant, poly (DL-lactide-coglycolide) microspheres, paraffin oil,squalene, virosome, AS03, AS04, IL-1, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, STING, Toll-like receptorligand, CD40L, pathogen-associated molecular patterns (PAMPs),damage-associated molecular pattern molecules (DAMPs), Freund's completeadjuvant, Freund's incomplete adjuvant, antibodies against immunesuppressive molecules (e.g., antibody or antagonist against transforminggrowth factor (TGF)-beta, A2aR antagonists), lipopolysaccharides (LPS),Fas ligand, Trail, lymphotactin, Mannan (M-FP), APG-2, Hsp70 and Hsp90.

In some embodiments, the lectin-bound pathogen construct comprises alectin, a portion of a lectin, an engineered lectin or a portionthereof. In some embodiments, the lectin is a collectin. In otherembodiments, the lectin is a ficollin. In some embodiments, the lectinis a mannose-binding lectin (MBL). In other embodiments, the lectincomprises amino acid residues 81 to 228 of MBL (SEQ ID NO: 1). In yetanother embodiment, the lectin comprises amino acid residues 111 to 228of MBL. In some embodiments, the mannose-binding lectin (MBL) is capableof binding to the pathogen. In some embodiments, the lectin comprisesSurfactant Protein D (SPD). In other embodiments, the Surfactant ProteinD (SPD) is capable of binding to the pathogen.

In some embodiments, the opsonin-bound or lectin-bound pathogenconstruct further comprises an immunoglobulin (IgG) Fc portion.

In some embodiments, the opsonin-bound or lectin-bound pathogenconstruct further comprises a solid substrate. In other embodiments, thesolid substrate is selected from the group consisting of a magneticbead, a microporous membrane, a hollow-fiber reactor, a blood filtrationmembrane and a blood flow device. In some embodiments, the solidsubstrate is a magnetic bead. In other embodiments, the pathogen ispresent on the solid substrate at a quantity of about 1 pg to about 1000μg.

In some embodiments, the pathogen is an infectious microorganismselected from the group consisting of a bacterium, a fungus, a virus anda parasite, or a fragment thereof.

In some embodiments, the bacterium is selected from the group consistingof Acinetobacter baumanii, Burkholderia cepacia, Bacterioides fragilis,Chlamydia trachomatis, Citrobacter freundii, Campylobacter jejuni,Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae,Haemophilus inf b, Helicobacter pylori, Klebsiella oxytoca, K. pneumonia(MDR/CRE), Legionella pneumophila, Neisseria meningitides, Neisseriagonorrhoeae, Pseudomonas aeruginosa, Salmonella typhi, paratyphi,typhimurium, Serratia marcescens, Shigella flexneri, Stenotrophomonasmaltophilia, Yersinia pseudotuberculosis, Bacillus subtilis, Clostridiumneoformans, C. difficile, C. perfringens, Corynebacterium spp,Enterococcus faecalis, Enterococcus faecium, vancomycin-resistantEnterococci (VRE), Listeria monocytogenes, Mycobactrium avium, M.tuberculosis, M. leprae, Nocardia farcinica, P. acnes, Staphylococcusaureus, methicillin-susceptible Staphylococcus aureus (MSSA),methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcusepidermidis, Streptococcus pyogenes, Strep Group A, Strep Group B(agalactiae) and Strep Group C.

In some embodiments, the bacterium is an antibiotic-resistant bacterium.In some embodiment, the bacterium is a multi-drug resistant bacterium.In other embodiments, the antibiotic-resistant bacterium or themulti-drug resistant bacterium is selected from the group consisting ofAcinetobacter baumanii, Escherichia coli, Klebsiella oxytoca, K.pneumonia (MDR/CRE), Pseudomonas aeruginosa, C. difficile,vancomycin-resistant Enterococci (VRE) and methicillin-resistantStaphylococcus aureus (MRSA).

In some embodiments, the fungus is selected from the group consisting ofAspergillus spp, Blastomyces, Candida albicans, glabrata,guilliermondii, krusei, parapsilosis, tropicalis Cryptococcus, Fusariumspp., Mucor spp., Saccharomyces, and Pneumocystis jirovecii (carinii).

In some embodiments, the virus is selected from the group consisting ofDengue virus, Ebola virus, EBV, Hepitis A virus, Hepitis B virus,Hepitis C virus, Hepitis D virus, HIV, HSV 1, HSV 2, Cytomegalovirus(CMV), Influenza A virus, Marburg virus, Human respiratory syncytialvirus (RSV), SARS-CoV, West Nile virus, Human papillomavirus (HPV),Human rhinoviruses (HRVs), and Zica virus.

In some embodiments, the parasite is selected from the group consistingof Cryptosporidium, Leishmania, Malaria, Schistosoma, Trichomonasm andTrypanosoma.

In some embodiments, the pathogen comprises a cell wall component of theinfectious microorganism. In other embodiments, the pathogen comprisesthe whole infectious microbial cell. In some embodiments, the cell wallcomponents of the infectious microorganism are glycosylated. In otherembodiments, the cell wall components are mannosylated. In someembodiments, the cell wall components are mannose-cappedlipoarabinomannan (ManLAM). In other embodiments, the cell wallcomponents are phosphatidylinositol mannoside (PIM).

In some embodiments, the pathogen is a mycoplasma. In other embodiments,the mycoplasma is selected from the group consisting of M. pneumoniae,M. hominis and M. orale.

In some embodiments, the pathogen comprises a pathogen-associatedmolecule pattern (PAMP). In other embodiments, the PAMP is selected fromthe group consisting of a pathogen fragment, a pathogen debris, apathogen nucleic acid, a pathogen lipoprotein, a pathogen surfaceglycoprotein, a pathogen membrane component, and a component releasedfrom the pathogen.

In some embodiments, the component released from the pathogen comprisesa toxin. In other embodiments, the toxin is selected from the groupconsisting of endotoxin, lipopolysaccharide (LPS), lipoteichoic acid(LTA), wall teichoic acid (WTA) and Ricin.

In some embodiments, the pathogen is in a sample derived from a subjectin vivo. In other embodiments, the sample is selected from the groupconsisting of a blood sample, a plasma sample, a serum sample, a bloodculture sample, a cerebrospinal fluid sample, a joint fluid sample, aurine sample, a semen sample, a saliva sample, a sputum sample, abronchial fluid sample, and a tear sample.

In some embodiments, the pathogen is derived from an in vitro culture, amicroorganism lysate, a crude lysate, or a purified lysate. In someembodiments, the pathogen is a synthetic pathogen.

In some embodiments, the pathogen is neutralized. In other embodiments,the pathogen is neutralized by treatment with antibiotics, ultravioletlight, sonication, microwave, bead mill, x-ray, autoclave, irradiationor mechanical disruption. In some embodiments, the pathogen isnon-infectious after neutralization.

In some embodiments, the immune cell is an antigen-presenting cell. Inother embodiments, the immune cell is selected from the group consistingof a dendritic cell, a macrophage, a T cell and a B cell.

In some embodiments, the vaccine composition comprises at least twodifferent types of pathogen. In other embodiments, the vaccinecomposition comprises at least three different types of pathogen. Insome embodiments, the vaccine composition is capable of targetingagainst different species of a pathogen.

In some embodiments, the vaccine composition is suitable forimplantation in a subject. In other embodiments, the vaccine compositionis suitable for subcutaneous implantation. In some embodiments, thevaccine composition is suitable for injection in a subject. In otherembodiments, the vaccine composition is suitable for oral administrationin a subject. In another embodiment, the vaccine composition is in theform of a pill, a tablet, a capsule, a soft gel, a chewable, a powder,an emulsion, or an aqueous solution.

In some embodiments, the vaccine composition is lyophilized. In otherembodiments, the vaccine composition has a shelf life of about 30 daysto about 1 year. In some embodiments, the vaccine composition has ashelf life of at least 1 year. In other embodiments, the vaccinecomposition is capable of being stored at room temperature. In someembodiments, the vaccine composition is portable.

In some embodiments, the subject is a mammal. In other embodiments, themammal is selected from the group consisting of a human, an embryo, ahorse, a dog, a cat, a cow, a sheep, a pig, a fish, an amphibian, areptile, a goat, a bird, a monkey, a mouse, a rabbit, and a rat. In someembodiments, the mammal is a human.

In some embodiments, the vaccine compositions further comprise ascaffold comprising a biomaterial and capable of recruiting andactivating the immune cell in the subject.

In some embodiments, the biomaterial is selected from the groupconsisting of glycosaminoglycan, silk, fibrin, MATRIGEL®,poly-ethyleneglycol (PEG), polyhydroxy ethyl methacrylate, polyvinylalcohol, polyacrylamide, poly (N-vinyl pyrolidone), poly(lactic acid),poly glycolic acid (PGA), poly lactic-co-glycolic acid (PLGA), polye-carpolactone (PCL), polyethylene oxide, poly propylene fumarate (PPF),poly acrylic acid (PAA), polyhydroxybutyric acid, hydrolysedpolyacrylonitrile, polymethacrylic acid, polyethylene amine, esters ofalginic acid; pectinic acid; and alginate, fully or partially oxidizedalginate, hyaluronic acid, carboxy methyl cellulose, heparin, heparinsulfate, chitosan, carboxymethyl chitosan, chitin, pullulan, gellan,xanthan, collagen, gelatin, carboxymethyl starch, carboxymethyl dextran,chondroitin sulfate, cationic guar, cationic starch, and combinationsthereof. In other embodiments, the biomaterial is selected from thegroup consisting of poly(L-lactide-co-glycolide) acid (PLGA), mesoporoussilica, cryogel, and combinations thereof.

In one aspect, the present invention provides methods of treating apathogen infection in a subject in need thereof. The methods compriseadministering the vaccine composition of the present invention to thesubject, thereby treating the pathogen infection in the subject.

In another aspect, the present invention provides methods of vaccinatinga subject against a pathogen infection. The methods compriseadministering the vaccine composition of the present invention to thesubject, thereby vaccinating the subject against the pathogen infection.

In one aspect, the present invention provides methods of treating anantibiotic-resistant bacterial infection in a subject in need thereof.The methods comprise administering the vaccine composition of thepresent invention to the subject, thereby treating theantibiotic-resistant bacterial infection in the subject. In someembodiments, the vaccine composition is specific for theantibiotic-resistant bacterium in the subject.

In another aspect, the present invention provides methods of decreasingthe level of a pathogen in a subject having a pathogen infection. Themethods comprise administering the vaccine composition of the presentinvention to the subject, thereby decreasing the level of the pathogenin the subject.

In some embodiments, the level of the pathogen is decreased in an organof the subject. In other embodiments, the organ is selected from thegroup consisting of a lung, a liver, a kidney, and a spleen.

In one aspect, the present invention provides methods of increasing thesurvival rate of a subject having a pathogen infection. The methodscomprise administering the vaccine composition of the present inventionto the subject, thereby increasing the survival rate of the subject.

In some embodiments, the subject is a mammal. In other embodiments, themammal is selected from the group consisting of a human, an embryo, ahorse, a dog, a cat, a cow, a sheep, a pig, a fish, an amphibian, areptile, a goat, a bird, a monkey, a mouse, a rabbit, and a rat. In someembodiments, the mammal is a human.

In some embodiments, the infection is an acute infection. In otherembodiments, the infection is a chronic infection.

In a further aspect, the present invention provides methods of producinga vaccine. The methods comprise contacting a sample comprising apathogen or fragment thereof with an opsonin or a lectin, wherein theopsonin or lectin is capable of binding to the pathogen or fragmentthereof in the sample, thereby forming an opsonin-bound or lectin-boundpathogen construct; isolating the opsonin-bound or lectin-bound pathogenconstruct from the sample; and combining the opsonin-bound orlectin-bound pathogen construct with a bioagent capable of recruiting animmune cell in a subject, thereby producing the vaccine.

In some embodiments, the pathogen is derived from a subject in vivo. Inother embodiments, the pathogen comprises a pathogen-associated moleculepattern (PAMP). In some embodiments, the PAMP is selected from the groupconsisting of a pathogen fragment, a pathogen debris, a pathogen nucleicacid, a pathogen lipoprotein, a pathogen surface glycoprotein, apathogen membrane component, and a released component from the pathogen.

In some embodiments, the pathogen is derived from an in vitro culture, amicroorganism lysate, a crude lysate, or a purified lysate. In otherembodiments, the pathogen is a synthetic pathogen.

In some embodiments, the bioagent comprises an adjuvant.

The present invention also provides vaccine compositions comprising ascaffold comprising a biomaterial and capable of recruiting andactivating an immune cell in a subject; and an opsonin-bound orlectin-bound pathogen construct.

In some embodiments, the biomaterial is selected from the groupconsisting of glycosaminoglycan, silk, fibrin, MATRIGEL®,poly-ethyleneglycol (PEG), polyhydroxy ethyl methacrylate, polyvinylalcohol, polyacrylamide, poly (N-vinyl pyrolidone), poly(lactic acid),poly glycolic acid (PGA), poly lactic-co-glycolic acid (PLGA), polye-carpolactone (PCL), polyethylene oxide, poly propylene fumarate (PPF),poly acrylic acid (PAA), polyhydroxybutyric acid, hydrolysedpolyacrylonitrile, polymethacrylic acid, polyethylene amine, esters ofalginic acid; pectinic acid; and alginate, fully or partially oxidizedalginate, hyaluronic acid, carboxy methyl cellulose, heparin, heparinsulfate, chitosan, carboxymethyl chitosan, chitin, pullulan, gellan,xanthan, collagen, gelatin, carboxymethyl starch, carboxymethyl dextran,chondroitin sulfate, cationic guar, cationic starch, and combinationsthereof. In other embodiments, the biomaterial is selected from thegroup consisting of poly(L-lactide-co-glycolide) acid (PLGA), mesoporoussilica, cryogel, and combinations thereof.

In some embodiments, the scaffold further comprises a bioagent. In otherembodiments, the bioagent is capable of recruiting the immune cell inthe subject.

In some embodiments, the bioagent is selected from the group consistingof interleukin (IL)-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10,IL-12, IL-15, IL-17, IL-18, tumor necrosis factor (TNF)-alpha,interferon (IFN)-gamma, IFN-alpha, granulocyte macrophage colonystimulating factor (GM-CSF), granulocyte colony stimulating factor(G-CSF), Fms-related tyrosine kinase ligand (FTL)-3 ligand, CCL19,CCL21, M-SCF, MIF, CD40L, CD3, ICAM, transforming growth factor(TGF)-beta, cytosine-guanosine oligonucleotide (CpG-ODN),lipopolysaccharides (LPS), Fas ligand, Trail, lymphotactin, Mannan(M-FP), APG-2, Hsp70 and Hsp90.

In some embodiments, the bioagent comprises an adjuvant. In otherembodiments, the adjuvant is selected from the group consisting ofcytosine-guanosine oligonucleotide (CpG-ODN) sequence, granulocytemacrophage colony stimulating factor (GM-CSF), ovalbumin (OVA),monophosphoryl lipid A (MPL), poly(I:C), MF59, alum, aluminum hydroxide,aluminum phosphate, calcium phosphate hydroxide, Quil A, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), FIA, montanide, adjuvant 65,lipovant, poly (DL-lactide-coglycolide) microspheres, paraffin oil,squalene, virosome, AS03, AS04, IL-1, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, STING, Toll-like receptorligand, CD40L, pathogen-associated molecular patterns (PAMPs),damage-associated molecular pattern molecules (DAMPs), Freund's completeadjuvant, Freund's incomplete adjuvant, antibodies against immunesuppressive molecules (e.g., antibody or antagonist against transforminggrowth factor (TGF)-beta, A2aR antagonists), lipopolysaccharides (LPS),Fas ligand, Trail, lymphotactin, Mannan (M-FP), APG-2, Hsp70 and Hsp90.

In some embodiments, the lectin-bound pathogen construct comprises alectin, a portion of a lectin, an engineered lectin or a portionthereof. In some embodiments, the lectin is a collectin. In otherembodiments, the lectin is a ficollin. In some embodiments, the lectinis a mannose-binding lectin (MBL). In other embodiments, the lectincomprises amino acid residues 81 to 228 of MBL (SEQ ID NO: 1). In yetanother embodiment, the lectin comprises amino acid residues 111 to 228of MBL. In some embodiments, the mannose-binding lectin (MBL) is capableof binding to the pathogen. In some embodiments, the lectin is SurfaceProtein D (SPD). In other embodiments, the Surface Protein D (SPD) iscapable of binding to the pathogen.

In some embodiments, the opsonin-bound or lectin-bound pathogenconstruct further comprises an immunoglobulin (IgG) Fc portion.

In some embodiments, the opsonin-bound or lectin-bound pathogenconstruct further comprises a solid substrate. In other embodiments, thesolid substrate is selected from the group consisting of a magneticbead, a microporous membrane, a hollow-fiber reactor, a blood filtrationmembrane and a blood flow device. In some embodiments, the solidsubstrate is a magnetic bead. In other embodiments, the pathogen ispresent on the solid substrate at a quantity of about 1 pg to about 1000μg.

In some embodiments, the pathogen is an infectious microorganismselected from the group consisting of a bacterium, a fungus, a virus anda parasite, or a fragment thereof.

In some embodiments, the bacterium is selected from the group consistingof Acinetobacter baumanii, Burkholderia cepacia, Bacterioides fragilis,Chlamydia trachomatis, Citrobacter freundii, Campylobacter jejuni,Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae,Haemophilus inf b, Helicobacter pylori, Klebsiella oxytoca, K. pneumonia(MDR/CRE), Legionella pneumophila, Neisseria meningitides, Neisseriagonorrhoeae, Pseudomonas aeruginosa, Salmonella typhi, paratyphi,typhimurium, Serratia marcescens, Shigella flexneri, Stenotrophomonasmaltophilia, Yersinia pseudotuberculosis, Bacillus subtilis, Clostridiumneoformans, C. difficile, C. perfringens, Corynebacterium spp,Enterococcus faecalis, Enterococcus faecium, vancomycin-resistantEnterococci (VRE), Listeria monocytogenes, Mycobactrium avium, M.tuberculosis, M. leprae, Nocardia farcinica, P. acnes, Staphylococcusaureus, methicillin-susceptible Staphylococcus aureus (MSSA),methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcusepidermidis, Streptococcus pyogenes, Strep Group A, Strep Group B(agalactiae) and Strep Group C.

In some embodiments, the bacterium is an antibiotic-resistant bacterium.In some embodiment, the bacterium is a multi-drug resistant bacterium.In other embodiments, the antibiotic-resistant bacterium or themulti-drug resistant bacterium is selected from the group consisting ofAcinetobacter baumanii, Escherichia coli, Klebsiella oxytoca, K.pneumonia (MDR/CRE), Pseudomonas aeruginosa, C. difficile,vancomycin-resistant Enterococci (VRE) and methicillin-resistantStaphylococcus aureus (MRSA).

In some embodiments, the fungus is selected from the group consisting ofAspergillus spp, Blastomyces, Candida albicans, glabrata,guilliermondii, krusei, parapsilosis, tropicalis Cryptococcus, Fusariumspp., Mucor spp., Saccharomyces, and Pneumocystis jirovecii (carinii).

In some embodiments, the virus is selected from the group consisting ofDengue virus, Ebola virus, EBV, Hepitis A virus, Hepitis B virus,Hepitis C virus, Hepitis D virus, HIV, HSV 1, HSV 2, Cytomegalovirus(CMV), Influenza A virus, Marburg virus, Human respiratory syncytialvirus (RSV), SARS-CoV, West Nile virus, Human papillomavirus (HPV),Human rhinoviruses (HRVs), and Zica virus.

In some embodiments, the parasite is selected from the group consistingof Cryptosporidium, Leishmania, Malaria, Schistosoma, Trichomonasm andTrypanosoma.

In some embodiments, the pathogen comprises a cell wall component of theinfectious microorganism. In other embodiments, the pathogen comprisesthe whole infectious microbial cell. In some embodiments, the cell wallcomponents of the infectious microorganism are glycosylated. In otherembodiments, the cell wall components are mannosylated. In someembodiments, the cell wall components are mannose-cappedlipoarabinomannan (ManLAM). In other embodiments, the cell wallcomponents are phosphatidylinositol mannoside (PIM).

In some embodiments, the pathogen comprises a cell wall component of theinfectious microorganism. In other embodiments, the pathogen comprisesthe whole infectious microbial cell. In some embodiments, the cell wallcomponents of the infectious microorganism are glycosylated. In otherembodiments, the cell wall components are mannosylated. In someembodiments, the cell wall components are mannose-cappedlipoarabinomannan (ManLAM). In other embodiments, the cell wallcomponents are phosphatidylinositol mannoside (PIM).

In some embodiments, the pathogen is a mycoplasma. In other embodiments,the mycoplasma is selected from the group consisting of M. pneumoniae,M. hominis and M. orale.

In some embodiments, the pathogen comprises a pathogen-associatedmolecule pattern (PAMP). In other embodiments, the PAMP is selected fromthe group consisting of a pathogen fragment, a pathogen debris, apathogen nucleic acid, a pathogen lipoprotein, a pathogen surfaceglycoprotein, a pathogen membrane component, and a component releasedfrom the pathogen.

In some embodiments, the component released from the pathogen comprisesa toxin. In other embodiments, the toxin is selected from the groupconsisting of endotoxin, lipopolysaccharide (LPS), lipoteichoic acid(LTA), wall teichoic acid (WTA) and Ricin.

In some embodiments, the pathogen is in a sample derived from a subjectin vivo. In other embodiments, the sample is selected from the groupconsisting of a blood sample, a plasma sample, a serum sample, a bloodculture sample, a cerebrospinal fluid sample, a joint fluid sample, aurine sample, a semen sample, a saliva sample, a sputum sample, abronchial fluid sample, and a tear sample.

In some embodiments, the pathogen is derived from an in vitro culture, amicroorganism lysate, a crude lysate, or a purified lysate. In someembodiments, the pathogen is a synthetic pathogen.

In some embodiments, the pathogen is neutralized. In other embodiments,the pathogen is neutralized by treatment with antibiotics, ultravioletlight, sonication, microwave, bead mill, x-ray, autoclave, irradiationor mechanical disruption. In some embodiments, the pathogen isnon-infectious after neutralization.

In some embodiments, the immune cell is an antigen-presenting cell. Inother embodiments, the immune cell is selected from the group consistingof a dendritic cell, a macrophage, a T cell and a B cell.

In some embodiments, the vaccine composition comprises at least twodifferent types of pathogen. In other embodiments, the vaccinecomposition comprises at least three different types of pathogen. Insome embodiments, the vaccine composition is capable of targetingagainst different species of a pathogen.

In some embodiments, the vaccine composition is suitable forimplantation in a subject. In other embodiments, the vaccine compositionis suitable for subcutaneous implantation. In some embodiments, thevaccine composition is suitable for injection in a subject. In otherembodiments, the vaccine composition is suitable for oral administrationin a subject. In another embodiment, the vaccine composition is in theform of a pill, a tablet, a capsule, a soft gel, a chewable, a powder,an emulsion, or an aqueous solution.

In some embodiments, the vaccine composition is lyophilized. In otherembodiments, the vaccine composition has a shelf life of about 30 daysto about 1 year. In some embodiments, the vaccine composition has ashelf life of at least 1 year. In other embodiments, the vaccinecomposition is capable of being stored at room temperature. In someembodiments, the vaccine composition is portable.

In some embodiments, the vaccine composition is capable of immobilizingthe opsonin-bound or lectin-bound pathogen construct and preventingleakage of the opsonin-bound or lectin-bound pathogen construct from thescaffold.

In some embodiments, the subject is a mammal. In other embodiments, themammal is selected from the group consisting of a human, an embryo, ahorse, a dog, a cat, a cow, a sheep, a pig, a fish, an amphibian, areptile, a goat, a bird, a monkey, a mouse, a rabbit, and a rat. In someembodiments, the mammal is a human.

In one aspect, the present invention provides stable scaffoldcompositions. The stable scaffold compositions comprise a biomaterialand capable of recruiting and activating an immune cell in a subject,wherein the scaffold is lyophilized, and wherein the scaffold has ashelf life of about 30 days to about 1 year.

In some embodiments, the biomaterial is selected from the groupconsisting of glycosaminoglycan, silk, fibrin, MATRIGEL®,poly-ethyleneglycol (PEG), polyhydroxy ethyl methacrylate, polyvinylalcohol, polyacrylamide, poly (N-vinyl pyrolidone), poly(lactic acid),poly glycolic acid (PGA), poly lactic-co-glycolic acid (PLGA), polye-carpolactone (PCL), polyethylene oxide, poly propylene fumarate (PPF),poly acrylic acid (PAA), polyhydroxybutyric acid, hydrolysedpolyacrylonitrile, polymethacrylic acid, polyethylene amine, esters ofalginic acid; pectinic acid; and alginate, fully or partially oxidizedalginate, hyaluronic acid, carboxy methyl cellulose, heparin, heparinsulfate, chitosan, carboxymethyl chitosan, chitin, pullulan, gellan,xanthan, collagen, gelatin, carboxymethyl starch, carboxymethyl dextran,chondroitin sulfate, cationic guar, cationic starch, and combinationsthereof. In other embodiments, the biomaterial is selected from thegroup consisting of poly(L-lactide-co-glycolide) acid (PLGA), mesoporoussilica, cryogel, and combinations thereof.

In some embodiments, the scaffold further comprises a bioagent. In someembodiments, the bioagent is capable of recruiting the immune cell inthe subject.

In some embodiments, the bioagent is selected from the group consistingof interleukin (IL)-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10,IL-12, IL-15, IL-17, IL-18, tumor necrosis factor (TNF)-alpha,interferon (IFN)-gamma, IFN-alpha, granulocyte macrophage colonystimulating factor (GM-CSF), granulocyte colony stimulating factor(G-CSF), Fms-related tyrosine kinase ligand (FTL)-3 ligand, CCL19,CCL21, M-SCF, MIF, CD40L, CD3, ICAM, transforming growth factor(TGF)-beta, cytosine-guanosine oligonucleotide (CpG-ODN),lipopolysaccharides (LPS), Fas ligand, Trail, lymphotactin, Mannan(M-FP), APG-2, Hsp70 and Hsp90.

In some embodiments, the bioagent comprises an adjuvant. In otherembodiments, the adjuvant is selected from the group consisting ofcytosine-guanosine oligonucleotide (CpG-ODN) sequence, granulocytemacrophage colony stimulating factor (GM-CSF), ovalbumin (OVA),monophosphoryl lipid A (MPL), poly(I:C), MF59, alum, aluminum hydroxide,aluminum phosphate, calcium phosphate hydroxide, Quil A, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), FIA, montanide, adjuvant 65,lipovant, poly (DL-lactide-coglycolide) microspheres, paraffin oil,squalene, virosome, AS03, AS04, IL-1, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, STING, Toll-like receptorligand, CD40L, pathogen-associated molecular patterns (PAMPs),damage-associated molecular pattern molecules (DAMPs), Freund's completeadjuvant, Freund's incomplete adjuvant, antibodies against immunesuppressive molecules (e.g., antibody or antagonist against transforminggrowth factor (TGF)-beta, A2aR antagonists), lipopolysaccharides (LPS),Fas ligand, Trail, lymphotactin, Mannan (M-FP), APG-2, Hsp70 and Hsp90.In certain embodiments, the adjuvant comprises granulocyte macrophagecolony stimulating factor (GM-CSF). In certain embodiments, the adjuvantcomprises a polyehylenimine (PEI)-CpG-ODN sequence.

In some embodiments, the immune cell is an antigen-presenting cell. Inother embodiments, the immune cell is selected from the group consistingof a dendritic cell, a macrophage, a T cell and a B cell.

In some embodiments, the scaffold composition is suitable forimplantation in a subject. In other embodiments, the scaffoldcomposition is suitable for subcutaneous implantation. In someembodiments, the scaffold composition is suitable for injection in asubject. In other embodiments, the scaffold composition is suitable fororal administration in a subject. In some embodiments, the scaffoldcomposition is in the form of a pill, a tablet, a capsule, a soft gel, achewable, a powder, an emulsion, or an aqueous solution.

In some embodiments, the subject is a mammal. In other embodiments, themammal is selected from the group consisting of a human, an embryo, ahorse, a dog, a cat, a cow, a sheep, a pig, a fish, an amphibian, areptile, a goat, a bird, a monkey, a mouse, a rabbit, and a rat. In someembodiments, the mammal is a human.

In some embodiments, the scaffold composition is capable of being storedat room temperature. In some embodiments, the scaffold composition isportable.

In another aspect, the present invention provides scaffold compositionscomprising a biomaterial and capable of recruiting and activating animmune cell in a subject, wherein the scaffold comprises a solidsubstrate, and wherein the solid substrate is suitable for attachment ofa pathogen.

In some embodiments, the solid substrate is selected from the groupconsisting of a magnetic bead, a microporous membrane, a hollow-fiberreactor, a blood filtration membrane and a blood flow device. In someembodiments, the solid substrate is a magnetic bead. In otherembodiments, the pathogen is present on the solid substrate at aquantity of about 1 pg to about 1000 μg.

In some embodiments, the biomaterial is selected from the groupconsisting of glycosaminoglycan, silk, fibrin, MATRIGEL®,poly-ethyleneglycol (PEG), polyhydroxy ethyl methacrylate, polyvinylalcohol, polyacrylamide, poly (N-vinyl pyrolidone), poly(lactic acid),poly glycolic acid (PGA), poly lactic-co-glycolic acid (PLGA), polye-carpolactone (PCL), polyethylene oxide, poly propylene fumarate (PPF),poly acrylic acid (PAA), polyhydroxybutyric acid, hydrolysedpolyacrylonitrile, polymethacrylic acid, polyethylene amine, esters ofalginic acid; pectinic acid; and alginate, fully or partially oxidizedalginate, hyaluronic acid, carboxy methyl cellulose, heparin, heparinsulfate, chitosan, carboxymethyl chitosan, chitin, pullulan, gellan,xanthan, collagen, gelatin, carboxymethyl starch, carboxymethyl dextran,chondroitin sulfate, cationic guar, cationic starch, and combinationsthereof. In other embodiments, the biomaterial is selected from thegroup consisting of poly(L-lactide-co-glycolide) acid (PLGA), mesoporoussilica, cryogel, and combinations thereof.

In some embodiments, the scaffold further comprises a bioagent. In someembodiments, the bioagent is capable of recruiting the immune cell inthe subject.

In some embodiments, the bioagent is selected from the group consistingof interleukin (IL)-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10,IL-12, IL-15, IL-17, IL-18, tumor necrosis factor (TNF)-alpha,interferon (IFN)-gamma, IFN-alpha, granulocyte macrophage colonystimulating factor (GM-CSF), granulocyte colony stimulating factor(G-CSF), Fms-related tyrosine kinase ligand (FTL)-3 ligand, CCL19,CCL21, M-SCF, MIF, CD40L, CD3, ICAM, transforming growth factor(TGF)-beta, cytosine-guanosine oligonucleotide (CpG-ODN),lipopolysaccharides (LPS), Fas ligand, Trail, lymphotactin, Mannan(M-FP), APG-2, Hsp70 and Hsp90.

In some embodiments, the bioagent comprises an adjuvant. In otherembodiments, the adjuvant is selected from the group consisting ofcytosine-guanosine oligonucleotide (CpG-ODN) sequence, granulocytemacrophage colony stimulating factor (GM-CSF), ovalbumin (OVA),monophosphoryl lipid A (MPL), poly(I:C), MF59, alum, aluminum hydroxide,aluminum phosphate, calcium phosphate hydroxide, Quil A, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), FIA, montanide, adjuvant 65,lipovant, poly (DL-lactide-coglycolide) microspheres, paraffin oil,squalene, virosome, AS03, AS04, IL-1, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, STING, Toll-like receptorligand, CD40L, pathogen-associated molecular patterns (PAMPs),damage-associated molecular pattern molecules (DAMPs), Freund's completeadjuvant, Freund's incomplete adjuvant, antibodies against immunesuppressive molecules (e.g., antibody or antagonist against transforminggrowth factor (TGF)-beta, A2aR antagonists), lipopolysaccharides (LPS),Fas ligand, Trail, lymphotactin, Mannan (M-FP), APG-2, Hsp70 and Hsp90.In certain embodiments, the adjuvant comprises granulocyte macrophagecolony stimulating factor (GM-CSF). In certain embodiments, the adjuvantcomprises a polyehylenimine (PEI)-CpG-ODN sequence.

In some embodiments, the immune cell is an antigen-presenting cell. Inother embodiments, the immune cell is selected from the group consistingof a dendritic cell, a macrophage, a T cell and a B cell.

In some embodiments, the scaffold composition is suitable forimplantation in a subject. In other embodiments, the scaffoldcomposition is suitable for subcutaneous implantation. In someembodiments, the scaffold composition is suitable for injection in asubject. In other embodiments, the scaffold composition is suitable fororal administration in a subject. In some embodiments, the scaffoldcomposition is in the form of a pill, a tablet, a capsule, a soft gel, achewable, a powder, an emulsion, or an aqueous solution.

In some embodiments, the subject is a mammal. In other embodiments, themammal is selected from the group consisting of a human, an embryo, ahorse, a dog, a cat, a cow, a sheep, a pig, a fish, an amphibian, areptile, a goat, a bird, a monkey, a mouse, a rabbit, and a rat. In someembodiments, the mammal is a human.

In some embodiments, the scaffold composition is lyophilized. In otherembodiments, the scaffold composition has a shelf life of about 30 daysto about 1 year. In some embodiments, the scaffold composition has ashelf life of at least 1 year. In other embodiments, the scaffoldcomposition is capable of being stored at room temperature. In someembodiments, the scaffold composition is portable.

In some embodiments, the pathogen is an infectious microorganismselected from the group consisting of a bacterium, a fungus, a virus anda parasite, or a fragment thereof.

In some embodiments, the pathogen comprises a pathogen-associatedmolecule pattern (PAMP). In other embodiments, the PAMP is selected fromthe group consisting of a pathogen fragment, a pathogen debris, apathogen nucleic acid, a pathogen lipoprotein, a pathogen surfaceglycoprotein, a pathogen membrane component, and a component releasedfrom the pathogen.

In some embodiments, the component released from the pathogen comprisesa toxin. In other embodiments, the toxin is selected from the groupconsisting of endotoxin, lipopolysaccharide (LPS), lipoteichoic acid(LTA), wall teichoic acid (WTA) and Ricin.

In some embodiments, the pathogen is in a sample derived from a subjectin vivo. In other embodiments, the sample is selected from the groupconsisting of a blood sample, a plasma sample, a serum sample, a bloodculture sample, a cerebrospinal fluid sample, a joint fluid sample, aurine sample, a semen sample, a saliva sample, a sputum sample, abronchial fluid sample, and a tear sample.

In some embodiments, the pathogen is derived from an in vitro culture, amicroorganism lysate, a crude lysate, or a purified lysate. In someembodiments, the pathogen is a synthetic pathogen.

In one aspect, the present invention provides a recombinant opsonin orlectin suitable for binding a pathogen or fragment thereof derived froma subject, wherein the opsonin or lectin comprises a pulmonarysurfactant. In some embodiments, the pulmonary surfactant is surfactantprotein D (SPD).

In another aspect, the present invention provides an opsonin-bound orlectin-bound pathogen constructs. The opsonin-bound or lectin-boundpathogen constructs comprise a pathogen or fragment thereof derived froma subject bound to an opsonin or a lectin, wherein the opsonin or lectincomprises a pulmonary surfactant. In some embodiments, the pulmonarysurfactant is surfactant protein D (SPD).

In another aspect, the present invention provides stable opsonin-boundor lectin-bound pathogen constructs. The stable opsonin-bound orlectin-bound pathogen constructs comprise a pathogen or fragment thereofderived from a subject bound to an opsonin, wherein the opsonin-bound orlectin-bound pathogen construct is lyophilized, and wherein theopsonin-bound or lectin-bound pathogen construct has a shelf life ofabout 30 days to about 1 year.

In some embodiments, the lectin-bound pathogen construct comprises alectin or a portion of a lectin, an engineered lection or a portionthereof. In some embodiments, the lectin is a collectin. In otherembodiments, the lectin is a ficollin. In some embodiments, the lectinis a mannose-binding lectin (MBL). In other embodiments, the lectincomprises amino acid residues 81 to 228 of MBL (SEQ ID NO: 1). In yetanother embodiment, the lectin comprises amino acid residues 111 to 228of MBL. In some embodiments, the mannose-binding lectin (MBL) is capableof binding to the pathogen. In some embodiments, the lectin is SurfaceProtein D (SPD). In other embodiments, the Surface Protein D (SPD) iscapable of binding to the pathogen.

In some embodiments, the opsonin-bound or lectin-bound pathogenconstruct further comprises an immunoglobulin (IgG) Fc portion.

In some embodiments, the opsonin-bound or lectin-bound pathogenconstruct further comprises a solid substrate. In other embodiments, thesolid substrate is selected from the group consisting of a magneticbead, a microporous membrane, a hollow-fiber reactor, a blood filtrationmembrane and a blood flow device. In some embodiments, the solidsubstrate is a magnetic bead. In other embodiments, the pathogen ispresent on the solid substrate at a quantity of about 1 pg to about 1000μg.

In some embodiments, the pathogen is an infectious microorganismselected from the group consisting of a bacterium, a fungus, a virus anda parasite, or a fragment thereof.

In some embodiments, the bacterium is selected from the group consistingof Acinetobacter baumanii, Burkholderia cepacia, Bacterioides fragilis,Chlamydia trachomatis, Citrobacter freundii, Campylobacter jejuni,Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae,Haemophilus inf b, Helicobacter pylori, Klebsiella oxytoca, K. pneumonia(MDR/CRE), Legionella pneumophila, Neisseria meningitides, Neisseriagonorrhoeae, Pseudomonas aeruginosa, Salmonella typhi, paratyphi,typhimurium, Serratia marcescens, Shigella flexneri, Stenotrophomonasmaltophilia, Yersinia pseudotuberculosis, Bacillus subtilis, Clostridiumneoformans, C. difficile, C. perfringens, Corynebacterium spp,Enterococcus faecalis, Enterococcus faecium, vancomycin-resistantEnterococci (VRE), Listeria monocytogenes, Mycobactrium avium, M.tuberculosis, M. leprae, Nocardia farcinica, P. acnes, Staphylococcusaureus, methicillin-susceptible Staphylococcus aureus (MSSA),methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcusepidermidis, Streptococcus pyogenes, Strep Group A, Strep Group B(agalactiae) and Strep Group C.

In some embodiments, the bacterium is an antibiotic-resistant bacterium.In some embodiment, the bacterium is a multi-drug resistant bacterium.In other embodiments, the antibiotic-resistant bacterium or themulti-drug resistant bacterium is selected from the group consisting ofAcinetobacter baumanii, Escherichia coli, Klebsiella oxytoca, K.pneumonia (MDR/CRE), Pseudomonas aeruginosa, C. difficile,vancomycin-resistant Enterococci (VRE) and methicillin-resistantStaphylococcus aureus (MRSA).

In some embodiments, the fungus is selected from the group consisting ofAspergillus spp, Blastomyces, Candida albicans, glabrata,guilliermondii, krusei, parapsilosis, tropicalis Cryptococcus, Fusariumspp., Mucor spp., Saccharomyces, and Pneumocystis jirovecii (carinii).

In some embodiments, the virus is selected from the group consisting ofDengue virus, Ebola virus, EBV, Hepitis A virus, Hepitis B virus,Hepitis C virus, Hepitis D virus, HIV, HSV 1, HSV 2, Cytomegalovirus(CMV), Influenza A virus, Marburg virus, Human respiratory syncytialvirus (RSV), SARS-CoV, West Nile virus, Human papillomavirus (HPV),Human rhinoviruses (HRVs), and Zica virus.

In some embodiments, the parasite is selected from the group consistingof Cryptosporidium, Leishmania, Malaria, Schistosoma, Trichomonasm andTrypanosoma.

In some embodiments, the pathogen is a mycoplasma. In other embodiments,the mycoplasma is selected from the group consisting of M. pneumoniae,M. hominis and M. orale.

In some embodiments, the pathogen comprises a cell wall component of theinfectious microorganism. In other embodiments, the pathogen comprisesthe whole infectious microbial cell. In some embodiments, the cell wallcomponents of the infectious microorganism are glycosylated. In otherembodiments, the cell wall components are mannosylated. In someembodiments, the cell wall components are mannose-cappedlipoarabinomannan (ManLAM). In other embodiments, the cell wallcomponents are phosphatidylinositol mannoside (PIM).

In some embodiments, the pathogen comprises a pathogen-associatedmolecule pattern (PAMP). In other embodiments, the PAMP is selected fromthe group consisting of a pathogen fragment, a pathogen debris, apathogen nucleic acid, a pathogen lipoprotein, a pathogen surfaceglycoprotein, a pathogen membrane component, and a component releasedfrom the pathogen.

In some embodiments, the component released from the pathogen comprisesa toxin. In other embodiments, the toxin is selected from the groupconsisting of endotoxin, lipopolysaccharide (LPS), lipoteichoic acid(LTA), wall teichoic acid (WTA) and Ricin.

In some embodiments, the pathogen is in a sample derived from a subjectin vivo. In other embodiments, the sample is selected from the groupconsisting of a blood sample, a plasma sample, a serum sample, a bloodculture sample, a cerebrospinal fluid sample, a joint fluid sample, aurine sample, a semen sample, a saliva sample, a sputum sample, abronchial fluid sample, and a tear sample.

In some embodiments, the pathogen is derived from an in vitro culture, amicroorganism lysate, a crude lysate, or a purified lysate. In someembodiments, the pathogen is a synthetic pathogen.

In some embodiments, the pathogen is neutralized. In other embodiments,the pathogen is neutralized by treatment with antibiotics, ultravioletlight, sonication, microwave, bead mill, x-ray, autoclave, irradiationor mechanical disruption. In some embodiments, the pathogen isnon-infectious after neutralization.

In some embodiments, the immune cell is an antigen-presenting cell. Inother embodiments, the immune cell is selected from the group consistingof a dendritic cell, a macrophage, a T cell and a B cell.

In some embodiments, the opsonin-bound or lectin-bound pathogenconstruct comprises at least two different types of pathogen. In otherembodiments, the opsonin-bound or lectin-bound pathogen constructcomprises at least three different types of pathogen. In someembodiments, the opsonin-bound or lectin-bound pathogen construct iscapable of targeting against different species of a pathogen.

In some embodiments, the opsonin-bound or lectin-bound pathogenconstruct is suitable for implantation in a subject. In otherembodiments, the opsonin-bound or lectin-bound pathogen construct issuitable for subcutaneous implantation. In some embodiments, theopsonin-bound or lectin-bound pathogen construct is suitable forinjection in a subject. In other embodiments, the opsonin-bound orlectin-bound pathogen construct is suitable for oral administration in asubject. In another embodiment, the vaccine composition is in the formof a pill, a tablet, a capsule, a soft gel, a chewable, a powder, anemulsion, or an aqueous solution.

In some embodiments, the opsonin-bound or lectin-bound pathogenconstruct is lyophilized. In other embodiments, the opsonin-bound orlectin-bound pathogen construct has a shelf life of about 30 days toabout 1 year. In some embodiments, the opsonin-bound or lectin-boundpathogen construct has a shelf life of at least 1 year. In otherembodiments, the opsonin-bound or lectin-bound pathogen construct iscapable of being stored at room temperature. In some embodiments, theopsonin-bound or lectin-bound pathogen construct is portable.

In some embodiments, the subject is a mammal. In other embodiments, themammal is selected from the group consisting of a human, an embryo, ahorse, a dog, a cat, a cow, a sheep, a pig, a fish, an amphibian, areptile, a goat, a bird, a monkey, a mouse, a rabbit, and a rat. In someembodiments, the mammal is a human.

The present invention also provides methods of treating a pathogeninfection in a subject in need thereof. The methods compriseadministering the vaccine composition of the present invention to thesubject, thereby treating the pathogen infection in the subject.

In one aspect, the present invention provides methods of vaccinating asubject against a pathogen infection. The methods comprise administeringthe vaccine composition of the present invention to the subject, therebyvaccinating the subject against the pathogen infection.

In another aspect, the present invention provides methods of treating anantibiotic-resistant bacterial infection in a subject in need thereof.The methods comprise administering the vaccine composition of thepresent invention to the subject, thereby treating theantibiotic-resistant bacterial infection in the subject. In someembodiments, the vaccine composition is specific for theantibiotic-resistant bacterium in the subject.

In one aspect, the present invention provides methods of decreasing thelevel of a pathogen in a subject having a pathogen infection. Themethods comprise administering the vaccine composition of the presentinvention to the subject, thereby decreasing the level of the pathogenin the subject.

In some embodiments, the level of the pathogen is decreased in an organof the subject. In some embodiments, the organ is selected from thegroup consisting of a lung, a liver, a kidney, and a spleen.

In one aspect, the present invention provides methods of increasing thesurvival rate of a subject having a pathogen infection. The methodscomprise administering the vaccine composition of the present inventionto the subject, thereby increasing the survival rate of the subject.

In another aspect, the present invention provides methods of reducingthe level of pain associated with a pathogen infection in a subject inneed thereof. The methods comprise administering the vaccine compositionof the present invention to the subject, thereby reducing the level ofpain associated with a pathogen infection in the subject.

In yet another aspect, the present invention provides methods ofreducing the level of distress associated with a pathogen infection in asubject in need thereof. The methods comprise administering the vaccinecomposition of the present invention to the subject, thereby reducingthe level of distress associated with a pathogen infection in thesubject.

In some embodiments, the subject is a mammal. In other embodiments, themammal is selected from the group consisting of a human, an embryo, ahorse, a dog, a cat, a cow, a sheep, a pig, a fish, an amphibian, areptile, a goat, a bird, a monkey, a mouse, a rabbit, and a rat. In someembodiments, the mammal is a human.

In some embodiments, the infection is an acute infection. In otherembodiments, the infection is a chronic infection.

In one aspect, the present invention provides methods of treating apathogen infection in a subject in need thereof. The methods compriseadministering the scaffold composition of the present invention and theopsonin-bound or lectin-bound pathogen construct of the presentinvention to the subject, thereby treating the pathogen infection in thesubject.

In another aspect, the present invention provides methods of vaccinatinga subject against a pathogen infection. The methods compriseadministering the scaffold composition of the present invention and theopsonin-bound or lectin-bound pathogen construct of the presentinvention to the subject, thereby vaccinating the subject against thepathogen infection.

In one aspect, the present invention provides methods of treating anantibiotic-resistant bacterial infection in a subject in need thereof.The methods comprise administering the scaffold composition of thepresent invention and the opsonin-bound or lectin-bound pathogenconstruct of the present invention to the subject, thereby treating theantibiotic-resistant bacterial infection in the subject. In someembodiments, the opsonin-bound or lectin-bound pathogen construct isspecific for the antibiotic-resistant bacterium in the subject.

In another aspect, the present invention provides methods of decreasingthe level of a pathogen in a subject having a pathogen infection. Themethods comprise administering the scaffold composition of the presentinvention and the opsonin-bound or lectin-bound pathogen construct ofthe present invention to the subject, thereby decreasing the level ofthe pathogen in the subject.

In some embodiments, the level of the pathogen is decreased in an organof the subject. In some embodiments, the organ is selected from thegroup consisting of a lung, a liver, a kidney, and a spleen.

In one aspect, the present invention provides methods of increasingsurvival rate of a subject having a pathogen infection. The methodscomprise administering the scaffold composition of the present inventionand the opsonin-bound or lectin-bound pathogen construct of the presentinvention to the subject, thereby increasing the survival rate of thesubject.

In another aspect, the present invention provides methods of reducingthe level of pain associated with a pathogen infection in a subject inneed thereof. The methods comprise administering the scaffoldcomposition of the present invention and the opsonin-bound orlectin-bound pathogen construct of the present invention to the subject,thereby reducing the level of pain associated with a pathogen infectionin the subject.

In yet another aspect, the present invention provides methods ofreducing the level of distress associated with a pathogen infection in asubject in need thereof. The methods comprise administering the scaffoldcomposition of the present invention and the opsonin-bound orlectin-bound pathogen construct of the present invention to the subject,thereby reducing the level of distress associated with a pathogeninfection in the subject.

In some embodiments, the scaffold composition of the present inventionand the opsonin-bound or lectin-bound pathogen construct of the presentinvention are administered simultaneously to the subject. In otherembodiments, the scaffold composition of the present invention isadministered to the subject prior to the opsonin-bound or lectin-boundpathogen construct of the present invention. In yet another embodiment,the scaffold composition of the present invention is administered to thesubject after the opsonin-bound or lectin-bound pathogen construct ofthe present invention.

In some embodiments, the subject is a mammal. In other embodiments, themammal is selected from the group consisting of a human, an embryo, ahorse, a dog, a cat, a cow, a sheep, a pig, a fish, an amphibian, areptile, a goat, a bird, a monkey, a mouse, a rabbit, and a rat. In someembodiments, the mammal is a human.

In some embodiments, the infection is an acute infection. In otherembodiments, the infection is a chronic infection.

In one aspect, the present invention provides methods of producing avaccine. The methods comprise contacting a sample comprising a pathogenor fragment thereof with an opsonin or a lectin, wherein the opsonin orthe lectin is capable of binding to a pathogen or fragment thereof inthe sample, thereby forming an opsonin-bound or lectin-bound pathogenconstruct; isolating the opsonin-bound or lectin-bound pathogenconstruct from the sample; and combining the isolated opsonin-bound orlectin-bound pathogen construct with a scaffold, thereby producing thevaccine.

In some embodiments, the pathogen is derived from a subject in vivo. Insome embodiments, the pathogen is derived from an in vitro culture, amicroorganism lysate, a crude lysate, or a purified lysate. In someembodiments, the pathogen is a synthetic pathogen.

In some embodiments, the pathogen comprises a pathogen-associatedmolecule pattern (PAMP). In some embodiments, the PAMP is selected fromthe group consisting of a pathogen fragment, a pathogen debris, apathogen nucleic acid, a pathogen lipoprotein, a pathogen surfaceglycoprotein, a pathogen membrane component, and a released componentfrom the pathogen.

In some embodiments, the pathogen comprises a cell wall component of theinfectious microorganism. In other embodiments, the pathogen comprisesthe whole infectious microbial cell. In some embodiments, the cell wallcomponents of the infectious microorganism are glycosylated. In otherembodiments, the cell wall components are mannosylated. In someembodiments, the cell wall components are mannose-cappedlipoarabinomannan (ManLAM). In other embodiments, the cell wallcomponents are phosphatidylinositol mannoside (PIM).

In another aspect, the present invention provides methods of producing avaccine. The methods comprise administering an opsonin or a lectin to asubject, wherein the opsonin or lectin is capable of binding to apathogen or fragment thereof, from the subject, thereby forming anopsonin-bound or lectin-bound pathogen construct; isolating theopsonin-bound or lectin-bound pathogen construct from the subject; andcombining the isolated opsonin-bound or lectin-bound pathogen constructwith a scaffold, thereby producing the vaccine.

In one aspect, the present invention provides kits for vaccinating asubject against a pathogen infection. The kits comprise a vaccinecomposition of the present invention; and instructions for administeringthe vaccine to the subject. In some embodiments, the vaccine compositionis prepackaged in a sterile container.

In another aspect, the present invention provides kits. The kitscomprise a scaffold composition of the present invention; anopsonin-bound or lectin-bound pathogen construct of the presentinvention, and instructions for administering the scaffold compositionand the opsonin-bound or lectin-bound pathogen construct to the subject.

In some embodiments, the scaffold composition and the opsonin-bound orlectin-bound pathogen construct are prepackaged in a sterile container.In other embodiments, the scaffold composition and the opsonin-bound orlectin-bound pathogen construct are prepackaged in different sterilecontainers. In certain embodiments, the scaffold composition and theopsonin-bound or lectin-bound pathogen construct are prepackaged in thesame sterile container.

The present invention is illustrated by the following drawings anddetailed description, which do not limit the scope of the inventiondescribed in the claims.

BRIEF DESCRIPTION THE DRAWINGS

FIG. 1 depicts the overall pathogen vaccine concept of the presentinvention.

FIG. 2 depicts FcMBL ELISA standard curve generated using the fungal MBLtarget, mannan. Specifically, 1 μM FcMBL coated supraparamagneticparticles were used to capture mannan in either buffer or whole donorblood. Serial dilutions of mannan was added to the indicated solutions,mixed with the FcMBL beads, assayed by ELISA and used to generate acurve for quantification of pathogen associated molecular patterns(PAMPs) from test samples.

FIG. 3 depicts the quantification of captured RS218 fragments on FcMBLbeads from titered antibiotics-treated RS218 solutions. The capturedRS218 fragments were quantified as pathogen associated molecularpatterns (PAMPs) using the standard curve generated by FcMBL ELISA.

FIG. 4 depicts the quantification by FcMBL ELISA of RS218 PAMPs capturedby FcMBL beads, and the specific calcium-dependent binding between FcMBLand RS218 PAMPs.

FIGS. 5A-5C depict images of a pathogen vaccine of the presentinvention. FIG. 5A depicts that the vaccine includes a PLG scaffoldcontaining FcMBL beads coated with captured antibiotics treated RS218pathogenic E. coli. FIG. 5B is an SEM image of FcMBL beads with capturedE. coli PAMPs incorporated into PLG scaffolds. The FcMBL Beads (1micron) are clearly visible dispersed throughout the holes and cavitiesin the PLG scaffolds. FIG. 5C is an SEM image of control scaffoldswithout the FcMBL beads.

FIG. 6 depicts the survival curves of vaccinated mice upon infectionwith a lethal dose of RS218 E. coli bacteria. Mice were implantedsubcutaneously with a PLG vaccine scaffold containing FcMBL capturedRS218 fragments for 3 weeks. Mice were infected intraperitoneally on day21 with a lethal dose of RS218. Survival of mice was monitored for 48hours and mice were humanely sacrificed earlier if clinical conditionsrequired. Vaccinated animals exhibited a significantly prolongedsurvival time. A prophylactic vaccine of PLG-GMCFS/CpG with FcMBL beadscoated with RS218 protected 9 out of 10 mice till the end of the studyat 48 hours while PLG scaffold alone or PLG with FcMBL beads/RS218lysate without recruiting and adjuvant factors did not protect.Treatment groups (n=10).

FIGS. 7A-7B depict the total organ pathogen counts and the individualorgan pathogen counts of vaccinated mice upon treatment with a lethaldose of RS218 bacteria, respectively. Mice were implanted subcutaneouslywith a PLG vaccine scaffold containing FcMBL captured RS218 fragmentsfor 3 weeks. Mice were infected intraperitoneally on day 21 with alethal dose of RS218. Organ cultures were collected in a sterilefashion, processed by mechanical disruption and plated to determine thetiter of pathogen in the organs. A significant reduction in pathogentiters was observed in vaccinated animals. Treatment groups (n=10).Pathogen loads were reduced by 2.5-3.5 logs (p=0.0021-0.0057).

FIG. 8 depicts the survival curves of mice vaccinated with PLG vaccinescaffolds containing either FcMBL captured RS218 fragments or wholeRS218 lysate. Mice were implanted subcutaneously with a PLG vaccinescaffold (with GM-CSF and CpG) or scaffolds containing either FcMBLcaptured RS218 fragments or whole RS218 lysate for 21 days, thenchallenged intraperitoneally with a sub-lethal dose of RS218 bacteria.Survival of vaccinated mice was monitored. Vaccinated animals exhibiteda significantly prolonged survival time.

FIG. 9 depicts the individual organ pathogen counts of mice vaccinatedwith PLG vaccine scaffolds containing either FcMBL captured RS218fragments or whole RS218 lysate. Mice were implanted subcutaneously witha PLG vaccine scaffold (with GM-CSF and CpG) or scaffolds containingeither FcMBL captured RS218 fragments or whole RS218 lysate for 21 days,then challenged intraperitoneally with a sub-lethal dose of RS218bacteria. Organ cultures were collected in a sterile fashion, processedby mechanical disruption and plated to determine the titer of pathogenin each individual organ. Organ cultures showed a significant reductionin pathogen titers in the vaccinated animals.

FIGS. 10A-10C depict in vitro quantification of the amount of CpG,GM-CSF and E. coli RS218 endotoxin leakage out of the PLG vaccinescaffolds, during leach of the sucrose, containing either FcMBL capturedRS218 fragments or whole RS218 lysate. No significant difference wasobserved for the CpG content (FIG. 10A) and the GM-CSF content (FIG.10B) when comparing between sham scaffolds, those with no beads (onlylysate), or beads with lysate. However, a significant endotoxin leakagewas observed in PLG vaccine scaffolds containing the whole RS218 lysate,whereas the PLG vaccine scaffolds containing the FcMBL captured RS218fragments demonstrated minimal leakage of endotoxin (FIG. 10C).

FIGS. 11A-11C are images taken at the sites of vaccine implantationdepicting the overall condition of the vaccinate mice. Mice wereimplanted subcutaneously with a PLG vaccine scaffold (with GM-CSF andCpG) or scaffolds containing either FcMBL captured RS218 fragments orwhole RS218 lysate for 21 days, then challenged intraperitoneally with asub-lethal dose of RS218 bacteria. Mice receiving the sham scaffolds hadno signs of immune reaction (FIG. 11A), and the vaccine scaffold waslargely intact in mice receiving the PLG vaccine scaffolds containingFcMBL captured RS218 fragments (FIG. 11B). However, mice receiving thePLG vaccine scaffolds containing whole RS218 lysate experiencedundesired side effects with formation of large abscesses (FIG. 11C).

FIGS. 12A-12B depict cross-reactivity of the infection vaccinetechnology. FIG. 12A depicts the survival curves of mice vaccinated withPLG vaccine scaffolds containing either FcMBL captured E. cloacae lysateor FcMBL captured E. coli RS218 lysate. Mice were implantedsubcutaneously with a PLG vaccine scaffold (with GM-CSF and CpG) orscaffolds containing either FcMBL captured E. cloacae lysate or FcMBLcaptured RS218 lysate for 21 days, then challenged intraperitoneallywith a lethal dose of RS218 bacteria. Survival of vaccinated mice wasmonitored. A prophylactic vaccine of PLG-GMCSF/CpG with FcMBL beadscoated with RS218 lysate protected 100% mice till the end of the studyat 96 hours from the RS218 challenge, and vaccine of PLG-GMCSF/CpG withFcMBL beads coated with E. cloacae lysate protected 78% mice till theend of the study at 96 hours from the RS218 challenge (LD90 at 20 hrs).PLG scaffolds with only GMCSF recruiting and CpG adjuvant (without FcMBLbeads and RS218 lysate) protected only 20% of animals at 96 hours. BothE. cloacae and E. coli are members of the order Enterobacteriaceae. FIG.12B depicts the individual organ pathogen counts of mice with PLGvaccine scaffolds containing either FcMBL captured E. cloacae lysate orFcMBL captured RS218 lsate. Organ cultures were collected in a sterilefashion, processed by mechanical disruption and plated to determine thetiter of pathogen in each individual organ. Organ cultures showed areduction in pathogen titers in the vaccinated animals.

FIG. 13 depicts a dose response study for vaccine compositions withdifferent scaffolds. Specifically, a prophylactic vaccine of MPSscaffold and GMCSF/CpG with FcMBL beads were coated with different dosesof E. coli RS218 Lysate. Vaccine with 15 PAMP units protected 90% ofmice from 21 days challenge while vaccine with 3 PAMP units onlyprotected 70% All mice receiving PLG vaccine scaffolds containingGMCSF/CpG and FcMBL beads coated with 15 PAMPs survived the challenge.

FIG. 14 depicts the survival curves of mice vaccinated with MPS vaccinescaffolds containing FcMBL captured RS218 lysate. A prophylactic vaccineof MPS-GMCSF/CpG with FcMBL beads coated with E. coli (RS218) lysateprotected 90% mice till the end of the study at 96 hours from the RS218challenge (n=12). Only 50% of mice vaccinated with sham MPS-GMCSF/CpGsurvived (n=6) with the RS218 challenge.

FIGS. 15A-15B depict the long-term effect of a single implanted dose ofthe infection vaccine technology. FIG. 15A depicts that a prophylacticvaccine of PLG-GMCSF/CpG with FcMBL beads coated with E. coli RS218lysate protected 100% of mice for more than 96 days despite (boosting)RS218 challenges at 21, 60 and 90 days. FIG. 15B depicts that the use ofa more challenged dose of RS218 bacteria, and shows that a prophylacticvaccine of PLG-GMCSF/CpG with FcMBL beads coated with E. coli RS218lysate protected 75% of mice for more than 96 days.

FIG. 16 are histology images depicting dense cellular infriltration withboth PLG and MPS scaffolds.

FIG. 17 depict the long-term antibody-mediated protection effect of theinfection vaccine technology, Specifically, FIG. 17 depicts a long-termIgG titer against the RS218 bacteria over a period of 90 days.

FIG. 18 depicts binding of FcMBL to Mycobacterium tuberculosis (MTb) intuberculosis and the potential use of the infection vaccine technologyfor treating tuberculosis. Specifically, FcMBL can bind to mannosylatedcomponents of Mycobacterium tuberculosis (MTb) cell wall e.g.mannose-capped Lipoarabinomannan (ManLAM), and PhosphatidylinositolMannoside (PIM).

FIG. 19 depicts the antibody-mediated effect of the infection vaccinetechnology against mannose-capped Lipoarabinomannan (ManLAM). When micewere vaccinated with a single dose of MPS-GMCSF/CpG containing FcMBLbeads coated with LAM Lysate, the titers of LAM-specific IgG increasedby 2-3 logs over pre-vaccinated naïve animals. (p=<0.001).

FIG. 20 depicts the cell-mediated anti-LAM response againstmannose-capped Lipoarabinomannan (ManLAM). Specifically, FIG. 20 depictsthe FACS analysis of infiltrating cells in sham MPS scaffolds or in MPSin PLG scaffolds containing FcMBL beads and MTb PAMPs. Control scaffolds(sham) showed few infiltrating cells, while dendritic cells (DCs),macrophages (Mphages) and CD4+ T cells were significantly increased intest scaffolds (Vax) (graph on right).

FIGS. 21A-21B depict the ability of FcMBL to capture multiple pathogengenera. FIG. 21A depicts that different amount of PAMPs from Gramnegative bacteria (E. coli RS218), Gram positive (MRSA, LAM), fungi(Candida albicans), viruses (HIV gp120) and parasites (Trichomonasvaginalis) were coated with FcMBL beads. FIG. 21B depicts an increasedantibody titer in mice on vaccination. Mice were vaccinated with asingle dose of MPS-GMCSF/CpG containing FcMBL beads coated with samplesfrom gram negative bacteria (E. coli RS218), Gram positive (MRSA, LAM),fungi (Candida albicans), viruses (HIV gp120) and parasites (Trichomonasvaginalis). The titers of LAM-specific IgG was increased overpre-vaccinated naïve animals in all cases.

FIG. 22 depicts the cell-mediated anti-Trichomonas response againstTrichomonas lysate incorporated into a vaccine. Specifically, FIG. 22depicts the FACS analysis of infiltrating cells in spleens of vaccinatedanimals. Control spleens (Naive) showed fewer infiltrating CD4+T cellsand CD11c cells than spleens in the vaccinated animal groups.

FIG. 23 depicts the antibody titers generated in mice vaccinated with avaccine composition comprising an alternative lectin, surfactant proteinD (SPD). Surfactant protein D (SPD) is another collectin (C-type lectinwith collagen region) related to MBL. An FcSPD was generated, which has77% protein sequence identity to FcMBL. FcSPD was shown to be able tobind the RS218 E. coli. An antibody-mediated immune reaction waselicited when FcSPD captured RS218 was incorporated into the MPSscaffold and used to vaccinate mice. An increased antibody titer wasobserved in vaccinated mice when compared to the non-vaccinated control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery thatpathogens or pathogen associated molecular patterns (PAMPs) isolatedusing an opsonin or lectin, e.g., an engineered lectin or fragmentthereof, can be used to generate functional vaccines for the treatmentof infectious diseases. In particular, the present inventors havesurprisingly discovered that pathogens or pathogen associated molecularpatterns (PAMPs) isolated using an engineered lectin, when combined witha bioactive agent, e.g., an adjuvant, and/or a scaffold, allow for therapid creation of high potency pathogen vaccines (FIG. 1). When used tovaccinate animals, a single dose of these vaccines resulted in asignificantly reduced pathogen titer in the vaccinated animals and asignificantly prolonged survival time after infecting the animals with alethal dose of bacteria. Indeed, as shown in Example 1, a single dose ofthe vaccine composition of the invention can protect the vaccinated micefrom bacteria challenge over a period of 90 days. In addition, theopsonin or lectin, e.g., an engineered lectin or fragment thereof, notonly functions to isolate a pathogen for use in the vaccine compositionsand present the pathogen to immune cells to initiate immune response,but also serves as an anchor structure to immobilize the pathogen, thuspreventing leakage of pathogen from the vaccine composition, andpreventing any undesired side effects currently experienced with thepathogen leakage.

The vaccine compositions of the present invention possess additionalimprovements over existing vaccines. For example, the vaccinecompositions of the present invention allow the rapid and directisolation of pathogens circulating in a blood sample from a patient withinfectious disease including both known and unknown pathogens, pathogenspresent within other biological fluids, or pathogens present in in vitrocultures. The claimed vaccine compositions can also be used againstpathogens that are difficult to isolate and purify. Once the pathogensare isolated from a subject, the vaccine compositions can be readilyprepared in a fast and convenient manner anywhere in the world, and canbe available for patients in a timely manner, for example, within oneday. In addition, vaccination using the claimed vaccine compositions canoccur in a more controlled, localized and safer manner, withoutcompromising the efficacy of the vaccine compositions. The improvedstability of the claimed vaccines allows them to be portable and be usedfor long term storage at room temperature without the need ofrefrigeration. Furthermore, the vaccine compositions can be multivalentvaccines when more than one type of pathogens are included in thecompositions, and can also be used to vaccinate against differentspecies or strains of a given pathogen. In addition, the vaccinecompositions, if implanted, can be easily removed from the subject aftervaccination. For example, in the case where too much immune response orundesired side effects are initiated after vaccination, the implantedvaccine compositions can be readily removed from the subject. Incontrast, current existing vaccines cannot be removed once they areinjected in the subjects. These improvements circumvent the majorlimitations of current pathogen vaccines, and would be of great interestto the public, especially during the time of an epidemic, for example,for populations in developing countries, or of great value for militaryuses, where vaccines that are readily available are highly desired.Indeed, the ability to rapidly create functional and highly stablevaccines that are not only easy for storage and handling, but may beadministered in a safer and more controlled manner and confer along-term protective effect, renders the vaccine compositions of thepresent invention significantly advantageous over existing vaccines.

Accordingly, the present invention provides vaccine compositions andmethods of producing such compositions. Other embodiments of theinvention include methods of treating a pathogen infection, methods ofvaccinating a subject against a pathogen infection, and methods fortreating an antibiotic-resistance bacterial infection in a subject inneed thereof. In further embodiments, the invention includes methods ofdecreasing the level of a pathogen in a subject having a pathogeninfection, methods of increasing the surviving rate of a subject havinga pathogen infection, methods of reducing pain associated with apathogen infection and methods of reducing distress associated with apathogen infection in a subject in need thereof. Novel scaffoldcompositions and pathogen compositions and uses thereof are alsoprovided herein.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso part of this invention.

In the following description, for purposes of explanation, specificnumbers, materials and configurations are set forth in order to providea thorough understanding of the invention. It will be apparent, however,to one having ordinary skill in the art that the invention may bepracticed without these specific details. In some instances, well-knownfeatures may be omitted or simplified so as not to obscure the presentinvention. Furthermore, reference in the specification to phrases suchas “one embodiment” or “an embodiment” means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the invention. The appearancesof phrases such as “in one embodiment” in various places in thespecification are not necessarily all referring to the same embodiment.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “comprising” or “comprises” is used herein in reference tocompositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

The term “vaccine,” as used herein, includes any composition containingan immunogenic determinant which stimulates the immune system such thatit can better respond to subsequent infections. A vaccine usuallycontains an immunogenic determinant, e.g., an antigen, and an adjuvant,the adjuvant serving to non-specifically enhance the immune response tothat immunogenic determinant. Currently produced vaccines predominantlyactivate the humoral immune system, i.e., the antibody dependent immuneresponse. Other vaccines focus on activating the cell-mediated immunesystem including cytotoxic T lymphocytes which are capable of killingtargeted pathogens.

The term “opsonin,” as used herein, refers to any protein or fragmentthereof that recognizes a surface molecule of a pathogen cell, e.g., apathogen associated molecular pattern (PAMP), thereby marking andtargeting the bound pathogen cell for destruction, e.g., by complementattack and phagocytosis. In some embodiments, the opsonin is a naturalopsonin, such as an antibody that is generated by B cells in response toantigen exposure, a complement protein that is part of the innate immuneresponse, or a soluble immune pattern-recognition protein that iscapable of identifying non-self or altered-self molecular patterns,coating the foreign microbes or altered/dying cells, and enhancingneutrophil reactivity against them by phagocytosis or complement attack(Litvack et al, 2010, Innate Immunity 16(3):191-200). Examples ofsoluble immune pattern-recognition proteins may include, but are notlimited to, collectins, ficolins, pentraxins, sCD14, MFG-E8, natural IgMand C1q.

The term “lectin,” as used herein, refers to any protein or fragmentthereof, that is capable of binding a carbohydrate structure, e.g., acarbohydrate structure on a pathogen or a carbohydrate structureassociated with a pathogen associated molecular pattern (PAMP). Lectinsinclude both naturally occurring lectins or engineered lectins, e.g.,engineered mannose binding lectins (MBL), surfactant protein D (SPD), orfragments thereof. For example, engineered mannose binding lectinsinclude the carbohydrate recognition domain of MBL, e.g., the neck andlectin domains of MBL, e.g., amino acid residues 81 to 228 of MBL oramino acid residues 111 to 228 of MBL, fused downstream to the Fcportion of human IgG.

The term “pathogen”, as used herein, refers to any pathogen or pathogenfragment capable of inducing an infectious disease in a subject. In someembodiments, the pathogen is an infectious microorganism selected fromthe group consisting of a bacterium, a fungus, a virus and a parasite,or a fragment thereof. The pathogen may comprise a whole infectiouspathogen cell, or a part of the pathogen cell, e.g., a cell wallcomponent of the infectious microorganism. In some embodiments, thepathogen comprises a pathogen-associated molecule pattern (PAMP), e.g.,a pathogen fragment, a pathogen debris, a pathogen nucleic acid, apathogen lipoprotein, a pathogen surface glycoprotein, a pathogenmembrane component, or a component released from the pathogen. The term“pathogen” also includes a mycoplasma or a toxin released from thepathogen. The pathogen for use in a vaccine composition of the presentinvention causes disease or infection in the species of a subject, e.g.,a mammal, to which the vaccine is administered, or in a closely relatedspecies. For example, the pathogen can be isolated from the same subjectwho receives the vaccine comprising the pathogen. Alternatively, thepathogen can be isolated from one subject having a pathogen infectionand the vaccine composition comprising the pathogen is administered to adifferent subject having the same pathogen infection. Pathogens suitablefor use in the vaccine compositions can also be derived from an in vitroculture, a microorganism lysate, a crude lysate, or a purified lysate,or alternatively, the pathogen may be a synthetic pathogen.

The term “pathogen associated molecular pattern” or “PAMP,” as usedherein, refers to any component of a microorganism that directs thetargeted host cell to distinguish “self” from “non-self”, e.g., infectedpathogen, and promotes signals associated with innate immunity.Exemplary PAMPs may include, but not limited to, a pathogen fragment; apathogen debris; a pathogen nucleic acid, including DNA (e.g.,unmethylated CpG motifs), double-stranded RNA (dsRNA), single-strandedRNA (ssRNA), and 5′-triphosphate RNA; a pathogen lipoprotein; a pathogensurface glycoprotein; a pathogen membrane component such aspeptidoglycans, glycosylphosphatidylinositol; and a component releasedfrom the pathogen, e.g., a toxin released from the pathogen. In someembodiments, the toxin is selected from the group consisting ofendotoxin, lipopolysaccharide (LPS), lipoteichoic acid (LTA), wallteichoic acid (WTA) and Ricin.

The term “bioagent”, as used herein, refers to any agent capable ofrecruiting an immune cell in a subject. The bioagent can be a naturallyoccurring, synthetically produced, or recombinantly produced compound. Abioagent suitable for use in the vaccine compositions of the claimedinvention include adjuvants; cytokines, e.g., IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, interferon-γ(γ-IFN), IFN-α, tumor necrosis factor (TNF); or growth factors, e.g.,transforming growth factor-α (TGF-α), TGF-β, granulocyte/macrophagecolony stimulating factor (GM-CSF), granulocyte colony stimulatingfactor (G-CSF), fibroblast growth factor (FGF), nerve growth factor(NFG), neurotrophins, epidermal growth factor (EGF), platelet derivedgrowth factor (PDGF), insulin-like growth factor (IGF), hepatocytegrowth factor (HGF), erythropoietin (EPO), thrombopoietin 9TPO),myostatin (GDF-8), growth differentiation factor-9 (GDF-9), acidicfibroblast growth factor (aFGF or FGF-1), or basic fibroblast growthfactor (bFGF or FGF-2).

The term “adjuvant”, as used herein, refers to compounds that can beadded to vaccines to stimulate immune responses against antigens.Adjuvants may enhance the immunogenicity of highly purified orrecombinant antigens. Adjuvants may reduce the amount of antigen or thenumber of immunizations needed to protective immunity. For example,adjuvants may activate antibody-secreting B cells to produce a higheramount of antibodies. Alternatively, adjuvants can act as a depot for anantigen, present the antigen over a longer period of time, which couldhelp maximize the immune response and provide a longer-lastingprotection. Adjuvants may also be used to enhance the efficacy of avaccine by helping to modify the immune response to particular types ofimmune system cells, for example, by activating T cells instead ofantibody-secreting B cells depending on the purpose of the vaccine.

The term “scaffold,” as used herein, refers to any structure comprisinga biomaterial and capable of recruiting and activating an immune cell ina subject. The scaffold provides a physical structure onto which or intowhich the opsonin- or lectin-bound pathogen construct or the bioagentcan associate or attach. The opsonin- or lectin-bound pathogen constructcan be encapsulated within the scaffold for delivery or administrationto a subject, therefore preventing any undesired leakage of pathogenfrom the vaccine composition. The scaffolds comprise a biocompatiblematerial that is not toxic or immunogenic. As used herein, the term“biocompatible material” refers to any material that does notdeteriorate appreciably and does not induce a significant immuneresponse or deleterious tissue reaction, e.g., toxic reaction orsignificant irritation, over time when implanted into or placed adjacentto the biological tissue of a subject, or induce blood clotting orcoagulation when it comes in contact with blood.

The term “immune cell,” as used herein, refers to any cell of the immunesystem that functions to protect the body against both infectiousdisease and foreign invaders. Exemplary immune cells include, but arenot limited to, a T cell, a B cell, an antigen-presenting cell, adendritic cell, a macrophage, a granulocytes, a monocyte, a neutrophil,and a natural killer (NK) cell. In some embodiments, the immune cell isan antigen-presenting cell.

The term “antigen-presenting cell,” as used herein, refers to any cellsthat are capable of displaying antigen on their surfaces.Antigen-presenting cells process antigens into peptide fragments andpresent them on the cell surface to the T cells of the immune system.Antigen-presenting cells can be found in a variety of tissue types.Classical antigen-presenting cells, including macrophages, B cells, anddendritic cells, associate with an MHC class II molecule to presentforeign antigens to helper T cells. While other cell types, e.g., mastcells, basophils, eosinophils, and group 3 innate lymphoid cells(ILC3s), that can express MHC class II molecules, may also serve asantigen-presenting cells (Kambayashi and Laufer, 2014, Nature ReviewsImmunology 14, 719-730). Antigen-presenting cells may also include anycells, e.g., nucleated cells, that are capable of displaying endogenouspeptides on the cell surface to cytotoxic T cells, typically using anMCH class I molecule. In addition to the MHC family of proteins, antigenpresentation relies on other specialized signaling molecules on thesurfaces of both APCs and T cells.

The term “lyophilization”, as used herein, refers to a dehydrationprocess used to preserve a composition, e.g., a vaccine composition, ascaffold composition and/or an opsonin-bound or lectin-bound pathogenconstruct of the present invention, or make the composition moreconvenient for storage and transport. Freeze-drying works by freezingthe composition and then reducing the surrounding pressure to allow thefrozen water in the composition to sublimate directly from the solidphase to the gas phase. In some embodiments, lyophilization increasesthe stability of the vaccine compositions of the claimed invention,thereby allows the vaccine compositions to be portable and to be storedat room temperature without the need of refrigeration.

By “treatment”, “prevention” or “amelioration” of a disease or disorder,e.g., an infectious disease caused by a pathogen, is meant delaying orpreventing the onset of such a disease or disorder, reversing,alleviating, ameliorating, inhibiting, slowing or stopping theprogression, aggravation or deterioration, the progression or severityof a condition associated with such a disease or disorder, e.g., apathogen infection. In one embodiment, the symptoms of a disease ordisorder, e.g., a pathogen infection, or pain and distress associatedwith a pathogen infection, are alleviated by at least 5%, at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 95%.

As used herein, a “subject” means a human or an animal. The animal maybe a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, sheep, pigs, goats, birds, horses, pigs, deer, bison,buffalo, amphibians, reptiles, feline species, e.g., domestic cat,canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu,ostrich, and fish, e.g., trout, catfish and salmon. In certainembodiments, the subject is an embryo or a fetus, where a life-longprotection is elicited after vaccination with the present invention.

In certain embodiments, the subject is a mammal, e.g., a primate, e.g.,a human. The terms, “patient” and “subject” are used interchangeablyherein. Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, pig, sheep, goat, bird,reptile, amphibian, fish or cow. Mammals other than humans can beadvantageously used as subjects that represent animal models ofinfectious diseases, or other related pathologies. A subject can be maleor female. The subject can be an adult, an adolescent or a child. Asubject can be one who has been previously diagnosed with or identifiedas suffering from or having a risk for developing an infectious disease,or disease and condition associated with pathogen infection.

II. Vaccine Compositions of the Invention

The invention provides vaccine compositions suitable for the prophylaxisand treatment of infectious diseases. The vaccine compositions comprisean opsonin- or lectin-bound pathogen construct and a bioagent capable ofrecruiting an immune cell in a subject.

The vaccine compositions of the present invention are tailored toactivate immune cells and prime the cells with a specific antigen, e.g.,a pathogen presented in the form of an opsonin-bound pathogen constructor in the form of a lectin-bound pathogen construct, thereby enhancingimmune defenses and destruction against the undesired pathogen. Thevaccine compositions attract appropriate immune cells, such asmacrophages, T cells, B cells, natural killer cells, and dendriticcells.

Any pathogen or pathogen fragment that is capable of inducing aninfectious disease in a subject may be used in the vaccine compositionsof the invention. In some embodiments, a pathogen is an infectiousmicroorganism, including both the whole infectious pathogen cell, or anycellular fragments of the infectious microorganism, e.g., cell wallcomponents of the infectious microorganism. In other embodiments, thepathogen comprises pathogen-released materials, pathogen debris,pathogen toxins, or pathogen-associated molecule patterns (PAMPs). Insome embodiments, the cell wall components of the infectiousmicroorganism are glycosylated. In other embodiments, the cell wallcomponents are mannosylated. In some embodiments, the cell wallcomponents are mannose-capped lipoarabinomannan (ManLAM). In otherembodiments, the cell wall components are phosphatidylinositol mannoside(PIM).

Examples of pathogens that are suitable for use in the vaccinecompositions include, but are not limited to, a bacterium, a virus, aviroid, a mycoplasma, a parasite, a fungus or a fragment thereof. Abacteria may be a member of the genus Neisseria, Aerobacter,Pseudomonas, Porphyromonas, Salmonella, Escherichia, Pasteurella,Shigella, Bacillus, Helibacter, Corynebacterium, Clostridium,Mycobacterium, Yersinia, Staphylococcus; Bordetelia, Brucelia, Vibrio,Streptococcus, Plasmodium, Schisostoma or Candida. Exemplary bacteriamay also include, but are not limited to, Acinetobacter baumanii,Burkholderia cepacia, Bacterioides fragilis, Chlamydia trachomatis,Citrobacter freundii, Campylobacter jejuni, Escherichia coli,Enterobacter aerogenes, Enterobacter cloacae, Haemophilus inf b,Helicobacter pylori, Klebsiella oxytoca, K. pneumonia (MDR/CRE),Legionella pneumophila, Neisseria meningitides, Neisseria gonorrhoeae,Pseudomonas aeruginosa, Salmonella typhi, paratyphi, typhimurium,Serratia marcescens, Shigella flexneri, Stenotrophomonas maltophilia,Yersinia pseudotuberculosis, Bacillus subtilis, Clostridium neoformans,C. difficile, C. perfringens, Corynebacterium spp, Enterococcusfaecalis, Enterococcus faecium, vancomycin-resistant Enterococci (VRE),Listeria monocytogenes, Mycobactrium avium, M. tuberculosis, M. leprae,Nocardia farcinica, P. acnes, Staphylococcus aureus,methicillin-susceptible Staphylococcus aureus (MSSA),methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcusepidermidis, Streptococcus pyogenes, Strep Group A, Strep Group B(agalactiae) and Strep Group C.

In some embodiments, the bacterium is an antibiotic-resistant bacterium.In some embodiment, the bacterium is a multi-drug resistant bacterium.In other embodiments, the antibiotic-resistant bacterium or themulti-drug resistant bacterium is selected from the group consisting ofAcinetobacter baumanii, Escherichia coli, Klebsiella oxytoca, K.pneumonia (MDR/CRE), Pseudomonas aeruginosa, C. difficile,vancomycin-resistant Enterococci (VRE) and methicillin-resistantStaphylococcus aureus (MRSA).

Any fungus may be used in the vaccine compositions of the presentinvention. Exemplary fungi may include, but are not limited to,Aspergillus spp, Blastomyces, Candida albicans, glabrata,guilliermondii, krusei, parapsilosis, tropicalis Cryptococcus, Fusariumspp., Mucor spp., Saccharomyces, and Pneumocystis jirovecii (carinii).

Exemplary viruses for use in the vaccine compositions of the presentinvention may include, but are not limited to, influenza virus, Denguevirus, Ebola virus, EBV, Hepitis A virus, Hepitis B virus, Hepitis Cvirus, Hepitis D virus, HSV 1, HSV 2, Cytomegalovirus (CMV), Influenza Avirus, Marburg virus, human respiratory syncytial virus (RSV), SARS-CoV,West Nile virus, human papillomavirus (HPV), human rhinoviruses (HRVs),Zica virus, human immunodeficiency virus (HIV-1 and HIV-2), human T-cellleukemia virus (HTLV), poliomyelitis virus, pox virus, measles virus,arbor virus, Coxsackie virus, herpes virus, hantavirus, Baculovirus,mumps virus, circovirus, vichaivirus, arenavirus, rotavirus,cytomegalovirus, avian leukosis-sarcoma virus (ALV), murine leukemiavirus (MLV), feline leukemia virus (FeLV), simian sarcoma virus (SIS),mouse mammary tumor virus (MMTV), Mason-Pfizer monkey virus (MPMV),simian AIDS viruses (SRVs), simian T-cell leukemia virus (SUN), bovineleukemia virus (BLV), simian immunodeficiency virus (SIV), felineimmunodeficiency virus (FTV), Visna/maedi virus (VMV), equine infectiousanemia virus (EIAV) and caprine arthritis-encephalitis virus (CAEV).

Exemplary parasites for use in the vaccine compositions of the presentinvention may include, but are not limited to, Cryptosporidium,Leishmania, Malaria, Schistosoma, Trichomonasm and Trypanosoma.Exemplary mycoplasma for use in the vaccine compositions of the presentinvention may include, but are not limited to, M. pneumoniae, M. hominisand M. orale.

In some embodiments, the pathogen comprises pathogen associatedmolecular patterns (PAMPs). The term “pathogen associated molecularpattern”, as used herein, refers to any component of a microorganismthat is recognized by cells of the innate immune system in the host(Tang et al., 2012, Immunol Rev., 249(1): 158-175 and Sangiuliano etal., 2014, Mediators of Inflammation, Article ID 821043). Specifically,since PAMPs are present in diverse organisms, but absent in the host,they provide exogenous danger signals, that are recognized by specificreceptors in host cells and alert the host immune system to the presenceof pathogens, thereby promoting immunity.

Exemplary PAMPs may include, but are not limited to, a pathogenfragment; a pathogen debris; a pathogen nucleic acid, including DNA(e.g., unmethylated CpG motifs), double-stranded RNA (dsRNA),single-stranded RNA (ssRNA), and 5′-triphosphate RNA; a pathogenlipoprotein; a pathogen surface glycoprotein; a pathogen membranecomponent such as peptidoglycans, glycosylphosphatidylinositol; and acomponent released from the pathogen, e.g., a toxin released from thepathogen. In some embodiments, the toxin is selected from the groupconsisting of endotoxin, lipopolysaccharide (LPS), lipoteichoic acid(LTA), wall teichoic acid (WTA) and Ricin.

PAMPs can be recognized by pattern recognition receptors (PPRs) such asToll-like receptors (TLRs) and other PRRs, such as retinoidacid-inducible gene I (RIG-I)-like receptors (RLRs), AIM2 like receptors(ALRs), and nucleotide-binding oligomerization domain (NOD)-likereceptors (NLRs). Following PAMP recognition, activated TLRs and otherPRRs localized to the cell surface, the cytoplasm, and/or intracellularvesicles provide signals to the host indicating the presence of amicrobial infection and trigger proinflammatory and antimicrobialresponses by activating a multitude of intracellular signaling pathways,including adapter molecules, kinases, and transcription factors such asnuclear factor-κB (NF-κB), activator protein-1 (AP-1), and IFNregulatory factors (IRFs). PAMP-induced signal transduction pathwaysultimately result in the activation of gene expression and the synthesisof a broad range of molecules, including cytokines, chemokines, celladhesion molecules, and immunoreceptors that direct the adaptive immuneresponse to invading pathogens by sensing microorganisms.

An effective amount of a pathogen or a pathogen fragment should beincluded in the vaccine composition. The effective amount of a pathogenor a pathogen fragment is the amount sufficient to produce an immuneresponse, e.g., an antibody secreting B cell or cytotoxic T cellmediated immune response, directed against one or more of the pathogenor pathogen fragments of the vaccine compositions of the invention. Theability of the vaccine compositions of the invention to elicit an immuneresponse can be determined using any routine method available to thoseof skill in the art. In some embodiments, the effective amount of eachcomposition is the amount sufficient to produce a cytotoxic T cellresponse in the subject as measured, for example, by a mixed lymphocyteT cell assay.

In some embodiments, the pathogens or pathogen fragments are present inthe vaccine composition at an amount of about 1 pg to about 1000 μg. Insome embodiments, the amount of pathogens or pathogen fragments is 1 μgto 100 μg, 1 μg to 200 μg, 1 μg to 300 μg, 1 μg to 400 μg, 1 μg to 500μg, 1 μg to 600 μg, 1 μg to 700 μg, 1 μg to 800 μg, or 1 μg to 900 μg.In some embodiments, the amount of pathogens or pathogen fragments is 1μg to 10 μg, 1 μg to 20 μg, 1 μg to 30 μg, 1 μg to 40 μg, 1 μg to 50 μg,1 μg to 60 μg, 1 μg to 70 μg, 1 μg to 80 μg, or 1 μg to 90 μg. In someembodiments, the amount of pathogens or pathogen fragments is 1 pg to100 μg, 1 pg to 90 μg, 1 pg to 80 μg, 1 pg to 70 μg, 1 pg to 60 μg, 1 pgto 50 μg, 1 pg to 40 μg, 1 pg to 30 μg, 1 pg to 20 μg or 1 pg to 10 μg.In other embodiments, the amount of pathogens or pathogen fragments is10 pg to 1 μg, 20 pg to 1 μg, 30 pg to 1 μg, 40 pg to 1 μg, 50 pg to 1μg, 60 pg to 1 μg, 70 pg to 1 μg, 80 pg to 1 μg, 90 pg to 1 μg, 100 pgto 1 μg, or 1000 pg to 1 μg.

Pathogens for use in the vaccine compositions of the present inventionare neutralized. For example, a pathogen is neutralized by treatmentwith antibiotics, ultraviolet light, sonication, microwave, bead mill,x-ray, autoclave, irradiation or mechanical disruption. Pathogens becomenon-infectious after neutralization. Use of non-infectious pathogens ina vaccine composition is beneficial and can reduce the potentiallysevere side effects experienced with pathogen toxins.

The vaccine compositions of the present invention may comprise one ormore types of pathogens to create a monovalent or multivalent vaccine.In some embodiments, the vaccine compositions comprise one type ofpathogen. In other embodiments, the vaccine compositions comprisemultiple types of pathogens, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or10 different types of pathogens, such as 2, 3, 4, 5, 6, 7, 8, 9, 10,1-4, 1-5, 2-5, 2-10, 3-6, 3-10 or 5-10 different types of pathogens.

The vaccine compositions of the present invention can also be used tovaccinate across species of a given pathogen. For example, the claimedvaccine compositions comprising one specific species or strain of apathogen can be used to vaccinate against multiple related species orstrains of the pathogen. The ability to vaccinate across species for agiven pathogen is particularly advantageous, especially for pathogensthat have multiple species or strains with different surface antigens.For those pathogens, inconsistency in the antigen structure between thevaccine strain and the infected strain would normally be a significantproblem. For example, when a pathogen strain, which is different fromthe vaccine strain, causes an infectious disease, the vaccination wouldbe rendered ineffective. However, this would not be a problem with theclaimed vaccine compositions given their superior ability to targetmultiple species of a particular pathogen.

Pathogens suitable for use in the vaccine compositions of the inventioncan be obtained from any sources. For example, a pathogen can beisolated directly from a sample derived from a subject in vivo. In someembodiments, the pathogen is isolated from the same subject who receivesthe vaccine comprising the pathogen. In other embodiments, the pathogenis isolated from one subject having a pathogen infection and the vaccinecomposition comprising the pathogen is administered to a differentsubject having the same or similar pathogen infection. For example, thepathogen can be isolated from a human subject, and administered to adifferent human subject infected with the same pathogen or relatedspecies of the pathogen. Alternatively, the pathogen can be isolatedfrom a subject, e.g., an animal, and administered to a differentsubject, e.g., a human, infected with the same pathogen or relatedspecies of the pathogen.

Examples of samples suitable for pathogen isolation include, but are notlimited to, a blood sample, a plasma sample, a serum sample, a bloodculture sample, a cerebrospinal fluid sample, a joint fluid sample, aurine sample, a semen sample, a saliva sample, a sputum sample, abronchial fluid sample, and a tear sample.

Alternatively, pathogens suitable for use in the vaccine compositionscan be derived from an in vitro culture, a microorganism lysate, a crudelysate, or a purified lysate. In another embodiment, the pathogen may bea synthetic pathogen.

Pathogens for use in the vaccine compositions are isolated using knownmethods in the art. In some embodiments, the pathogen is isolated from asample using an opsonin or a lectin and presented in the form of anopsonin-bound pathogen construct or a lectin-bound pathogen construct ina vaccine composition.

As demonstrated in Example 1, pathogen fragments isolated using a lectindemonstrate an unexpected advantage over the direct use of amicroorganism lysate, e.g., a bacterial lysate. Although both thebacterial lysate and the lectin captured bacterial fragments may be usedto generate functional vaccine compositions and protect mice from alethal challenge of bacterial infection, the direct use of a bacteriallysate in a vaccine composition was associated with undesirable sideeffects such as leaching of the lysate and formation of abscesses at thesite of administration. Therefore, the lectin or opsonin, not onlyfunctions to isolate a pathogen for use in the vaccine compositions andpresent the pathogen to immune cells in order to elicitate long-termimmune response, but also serves as an anchor structure to immobilizethe pathogen, thereby preventing leakage of the pathogen from thevaccine composition.

Direct isolation of a pathogen using a lectin or an opsonin confersadditional benefit, for example, when a particular pathogen or pathogenfragment, e.g., LPS, is difficult to purify, or when a pathogen isunknown or hard to culture, addition of the lectin or opsonin into amicroorganism lysate would facilitate the isolation of a desiredpathogen, thereby forming the opsonin- or lectin-bound pathogenconstruct that can be used in the vaccine compositions.

An opsonin suitable for use in the vaccine compositions of the presentinvention includes any protein or fragment thereof, that can recognize asurface molecule of a pathogen cell, e.g., a pathogen associatedmolecular pattern, thereby targeting the opsonin-bound pathogen cell fordestruction by complement attack and phagocytosis.

In some embodiments, the opsonin in the opsonin-bound pathogen constructis a natural opsonin or a portion thereof, such as an antibody that isgenerated by B cells in response to antigen exposure, a complementprotein that is part of the innate immune response, or a soluble immunepattern-recognition protein that is capable of identifying non-self oraltered-self molecular patterns, coating the foreign microbes oraltered/dying cells, and enhancing neutrophil reactivity against them byphagocytosis or complement attack (Litvack et al, 2010, Innate Immunity16(3):191-200). Typically, foreign pathogens display altered arrays ofmolecules on their surfaces. For example, cell surfaces of foreignmicrobes often contain, or allow the access to different lipids,intracellular glycoproteins and nucleic acids such as DNA than thosefrom host cells. These cell surface patterns are considered as signalsfor opsonin molecules, which will mark and target the recognized cellsfor killing. Similarly, a programmed cell death process such asapoptosis also generates cell surface blebs that contain intracellularcomponents. These blebs are easily released for effective clearance orsignaling. During late stages of cell death, soluble components such aslysoPC and nucleotides are also released that act as signals for opsoninmolecules. (Litvack et al, 2010, Innate Immunity 16(3):191-200).Examples of soluble immune pattern-recognition protein may include, butnot limited to, collectins, ficolins, pentraxins, sCD14, MFG-E8, naturalIgM and C1q which can effectively identify some of these specificmolecular patterns.

Engineered opsonins or lectins, e.g., engineered mannose bindinglectins, surfactant protein D, or fragments thereof, may also be used inthe vaccine compositions of the present invention. With the use ofgenetic engineering, and direct evolution and selection strategies,modified versions of natural opsonins or lectins can be engineered. Suchengineered opsonin or lectin molecules may be used to bind pathogens oridentify specific pathogen species for use in the vaccine compositionfor treatment and diagnosis of patients with infectious diseases.

In some embodiments, the opsonin-bound or lectin-bound pathogenconstruct comprises a lectin, a portion of a lectin, an engineeredlectin or a portion thereof. In some embodiments, the lectin is acollecin. In other embodiments, the lectin is a ficolin.

In some embodiments, the lectin comprises pulmonary surfactant protein D(SPD). In other embodiments, the surfactant protein D (SPD) is capableof binding to the pathogen. Pulmonary surfactant, a complex mixture oflipids and proteins, is essential for lung function. Pulmonarysurfactant protein D (SPD) has a significant role in immune andinflammatory regulation of the lung as it regulates of the level ofsurfactant in the lungs and acts as a host defense protein.

In some embodiments, the lectin is a mannose-binding lectin (MBL). MBLis a serum lectin that binds to mannose, N-acetylglucos amine(NAG)-containing carbohydrates, and various other carbohydrates that arepresent on the surface of many microbial pathogens. MBL (also calledmannose- or mannan-binding protein, MBP) is a polymeric proteinassembled from three or more 32 kDa monomers. Each monomer has anN-terminal cysteine rich region, a collagen-like gly-X-Y region, a neckregion and a carbohydrate recognition domain. The assembly of the highermolecular weight polymers begins with formation of trimers of the 32 kDamonomer; these trimers then self-assembly into higher molecular weightpolymers of three to six sets of trimers.

MBL is a key component in opsonization of microbial pathogens and in theactivation of complement (via the lectin pathway) and coagulation.Opsonization is the binding of proteins to target cells and thentargeting these cells for uptake and destruction by phagocytic cells,such as macrophages and neutrophils. This opsonization appears to bemediated by the small, cysteine-rich N-terminal domain of MBL as well asC3b deposited on the target cell surface by MBL-mediated lectincomplement pathway activation. In the activation of complement via thelectin pathway, the microbe and specialized proteins, i.e., MASP-1(Mannan-binding lectin Associated Serine Protease) and MASP-2, interactwith bound MBL and activate complement in the absence of antibody(Matsushita & Fujita, 1992, J. Exp. Med. 176(5): 1497-1502; Thiel etal., 1997, Nature 386: 506-510). The higher molecular weight MBLcomplexes (5 to 6 repeats of the functional MBL trimer) are potentactivators of complement via this lectin pathway, in which MASP 2appears to activate complement, and MASP 1 activates coagulation. Thesmaller complexes (three to four repeats of the MBL trimer unit) are themost potent activators of coagulation (Krarup et al., 2007 PLoS One,2(7), e623).

MBL is an excellent choice for use in the vaccine compositions describedherein; however, the intact molecule is not typically used in thepresence of whole blood because the wild type MBL has multiplefunctional domains that can activate phagocytosis, blood coagulation andcomplements, which could interfere with or complicate therapeuticvaccine function. This characteristic of MBL can be separated from itspathogen binding function as provided herein. More specifically, MBLcontains four parts, from N- to C-terminus: a small N-terminal domain ofessentially unknown function that may be involved in macrophage bindingand/or MASP binding; a collagen segment that may also be involved inMASP binding and higher-order oligomerization; an alpha-helical “neck”segment that is sufficient for trimerization; and the carbohydraterecognition lectin domain at the C-terminus that mediates directpathogen binding. The lectin domain is useful for the present inventionfor isolation of pathogens. In some embodiments, the lectin comprisesthe neck and lectin domains of MBL. For example, the lectin may compriseamino acid residues 81 to 228 of MBL (SEQ ID NO: 1), or amino acidresidues 111 to 228 of MBL. See, e.g., U.S. Pat. No. 9,150,631. Theentire contents of the foregoing patent are incorporated herein byreference.

Amino acid residues 81-228 of human MBL (which includes the coiled coilneck region and the carbohydrate recognition domains (CRD) of human MBL:81 pdgdsslaas erkalqtema rikkwltfsl gkqvgnkffl tngeimtfek vkalcvkfqa 141svatprnaae ngaiqnlike eaflgitdek tegqfvdltg nrltytnwne gepnnagsde 201dcvlllkngq wndvpcstsh lavcefpi (SEQ ID NO: 1).

The binding characteristics of a lectin, e.g., MBL, can be manipulatedby directed evolution for altered binding specificity. MBL may bemodified so that it binds to a limited set of sugars or other molecularfeatures, such that the modified MBL will bind to a selected set ofmicrobes to provide a more specific capability for pathogen classcapture. For example, the engineered MBL may be capable of capturecertain pathogen class, e.g., one or more bacterial species, e.g., gramnegative bacteria or gram positive bacteria; one or more virusesspecies; one or more fungi species; or one or more protozoan species.

For example, the engineered lectin, e.g., MBL, for use in the vaccinecompositions of the present invention may be generated by looking at anatomic structure of MBL complexed with a sugar, and then mutatingappropriate amino acids that make contact in a sugar-specific manner,such that distinctive contacts are lost or particular types of sterichindrance are created. For example, the three dimensional structure ofrat MBL has been solved in a complex with a high-mannoseoligosaccharide, with N-acetylglucosamine, and with a methylated fucose.His189Val and Ile207Val are examples of substitutions that modificationsalter specificity.

A directed evolution strategy can be applied to MBL in order to selectMBL variants with specific binding to a specific type of pathogen, forexample, yeast, gram-positive bacteria, gram-negative, coagulasenegative, and aerobic bacteria. Derivatives of MBL with a particularspecificity can be isolated by any method known in the art, for example,a standard phage display strategy. First, a set of MBL variants can beexpressed from a phagemid vector; then binds this library to a target ofinterest (e.g., E. coli) and one or two rounds of selection may beperformed. Subsequently, a round of negative selection against a relatedtarget (e.g., Candida) is followed, taking those phagemids that fail tobind. These cycles of positive and negative selection are then repeateduntil a population of phages that generally bind to the target and donot bind to the non-target is generated. This method may be applied toany pair of microbial strains against which differential binding isdesired, such as bacteria that are resistant and sensitive to a givenantibiotic.

MBL belongs to the class of collectins in the C-type (calcium-dependent)lectin superfamily, other members of which, such as surfactant proteinA, surfactant protein D, CL-L1 and CL-P1, may be useful for constructionof engineered lectins for use in the vaccine compositions of the presentinvention. Other possible lectins include ficollin which can alsoactivate the lectin pathway of complement and bind MASP proteins.Ficollin proteins are related to MBL but have a different, and a morelimited specificity. In the context of the vaccine compositionsdescribed herein, one option is to use the lectin domain of a ficollinthat corresponds to the lectin domain of MBL described above. Anotherapproach is to use ‘shuffling’ of segments or individual amino acidsbetween MBL and one or more Ficollins to create hybrid molecules thatmay have hybrid specificities. The directed evolution and selectionapproach described above also could potentially be used to generateengineered proteins that provide the class, subclass and speciesspecificity.

The opsonin or lectin, e.g., engineered MBL or SPD, may further comprisean immunoglobulin Fc portion. For example, the neck and lectin domainsof MBL may be fused downstream to the Fc portion of human IgG. The Fcportion may include the CH2-CH3 interface of the IgG Fc domain, whichcontains the binding sites for a number of Fc receptors includingStaphylococcal protein A. In use, the Fc portion dimerizes andstrengthens the avidity affinity of the binding by MBL lectins tomonomeric sugars. Additionally, the n-linked glycosylation of therecombinant lectin can be removed. For example, when the neck and lectindomains of MBL, e.g., amino acid residues 81-228 of MBL, are fused withthe Fc portion of IgG, the glycosylation of the resulting fusion proteincan be removed by changing the amino acid at residue 297 from asparagineto aspartic acid (N297D) in the Kabat system of numbering amino acids inantibodies, which corresponds to amino acid 82 in this particular FcMBLconstruct.

The opsonin or lectin, e.g., engineered MBL or SPD, may further comprisea solid substrate. Any solid substrate that is for attachment of thefusion MBL lectin-Fc or SPD-Fc protein can be used in the vaccinecomposition of the present invention. The solid substrate may facilitatea long-term presentation of pathogen to an immune cell. Examples ofsolid substrates may include, but not limited to, a bead, e.g., amagnetic bead, a microporous membrane, a hollow-fiber reactor, a bloodfiltration membrane and a blood flow device.

In some embodiments, the solid substrate may comprise beads, e.g.,magnetic beads, or other structured materials, which then pull pathogensout from a sample (including biological fluids such as blood or in vitropathogen cultures, and any other samples as described above), andconcentrate and collect the pathogens, including living pathogens.Alternatively, the solid substrate may comprise a hollow-fiber reactoror any other blood filtration membrane or flow device (e.g., a simpledialysis tube) or other resins, fibers, or sheets to selective bind andsequester the biological pathogens.

The beads, e.g., magnetic beads, can be of any shape, including but notlimited to spherical, rod, elliptical, cylindrical, disc, and the like.In some embodiments, beads, e.g., magnetic beads, having a truespherical shape and defined surface chemistry are used to minimizechemical agglutination and non-specific binding. As used herein, theterm “magnetic bead” refers to a nano- or micro-scale particle that isattracted or repelled by a magnetic field gradient or has a non-zeromagnetic susceptibility. The magnetic bead can be paramagnetic orsuper-paramagnetic. In some embodiments, magnetic beads aresuper-paramagnetic. Magnetic beads are also referred to as magneticparticles. In some embodiments, magnetic beads having a polymer shellare used to protect the pathogen from exposure to iron. For example,polymer-coated magnetic beads can be used to protect pathogens fromexposure to iron.

The beads, e.g., magnetic beads, can range in diameter from 1 nm to 1mm. For example, beads, e.g., magnetic beads, are about 50 nm indiameter, about 128 nm in diameter, about 500 nm in diameter, about 1 μmin diameter, about 250 nm to about 250 μm in diameter, 0.1 μm to 100 μmin diameter, 0.1 μm to 50 μm in diameter, or 0.1 μm to 10 μm indiameter. In some embodiments, the magnetic bead is a magneticnano-particle or magnetic micro-particle. Magnetic nanoparticles are aclass of nanoparticle which can be manipulated using magnetic field ormagnetic field gradient. Such particles commonly consist of magneticelements such as iron, nickel or cobalt. Magnetic nano-particles arewell-known and methods for their preparation have been described in theart. See, e.g., U.S. Pat. Nos. 6,878,445; 5,543,158; 5,578,325;6,676,729; 6,045,925; and 7,462,446; and U.S. Patent Publications No.2005/0025971; No. 2005/0200438; No. 2005/0201941; No. 2005/0271745; No.2006/0228551; No. 2006/0233712; No. 2007/01666232; and No. 2007/0264199.The entire contents of the foregoing patents and patent applications areincorporated herein by reference.

Magnetic beads for use in the vaccine compositions of the presentinvention are easily and widely available commercially, with or withoutfunctional groups capable of binding to affinity molecules. Suitablemagnetic beads are commercially available such as from Dynal Inc. (LakeSuccess, N.Y.); PerSeptive Diagnostics, Inc. (Cambridge, Mass.);Invitrogen Corp. (Carlsbad, Calif.); Cortex Biochem Inc. (San Leandro,Calif.); and Bangs Laboratories (Fishers, Ind.). In particularembodiments, magnetic particles are MyOne™ Dynabeads® magnetic beads(Dynal Inc.).

The opsonin or lectin, e.g., engineered opsonin or lectin, can beconjugated with a solid substrate by methods well known in the art forconjugating peptides with other molecules. For example, Hermanson,Bioconjugate Techniques (2nd Ed., Academic Press (2008)) and Niemeyr,Bioconjugation Protocols: Strategies & Methods, in Methods In MolecularBiology (Humana Press, 2004), provide a number of methods and techniquesfor conjugating peptides to other molecules.

Alternatively, the surface of the solid substrate can be functionalizedto include binding molecules that bind selectively with the opsonin orlectin. These binding molecules are also referred to as affinitymolecules. The binding molecule can be bound covalently ornon-covalently on the surface of the solid substrate. As used herein,the term “binding molecule” or “affinity molecule” refers to anymolecule that is capable of specifically binding an engineered opsoninor lectin described herein. Representative examples of binding moleculesinclude, but are not limited to, antibodies, antigens, proteins,peptides, nucleic acids (DNA, RNA, PNA and nucleic acids that aremixtures thereof or that include nucleotide derivatives or analogs);receptor molecules, such as the insulin receptor; ligands for receptors(e.g., insulin for the insulin receptor); and biological, chemical,polymeric or other molecules that have affinity for another molecule,such as biotin and avidin.

Binding molecules can be conjugated to surface of the solid substrateusing any of a variety of methods known to those of skill in the art.The binding molecule can be coupled or conjugated to surface of thesolid substrate covalently or non-covalently. The covalent linkagebetween the binding molecule and the surface can also be mediated by alinker. The non-covalent linkage between the binding molecule and thesurface can be based on ionic interactions, van der Waals interactions,dipole-dipole interactions, hydrogen bonds, electrostatic interactions,and/or shape recognition interactions.

The vaccine compositions of the present invention comprise one or morebioagents. As used herein, the term “bioagent” refers to any agentcapable of recruiting an immune cell in a subject. The bioagent can benaturally occurring, synthetically produced, or recombinant compounds. Abioagent suitable for use in the vaccine compositions of the claimedinvention include adjuvants; cytokines, e.g., IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, interferon-γ(γ-IFN), IFN-α, tumor necrosis factor (TNF); or growth factors, e.g.,transforming growth factor-α (TGF-α), TGF-β, granulocyte/macrophagecolony stimulating factor (GM-CSF), granulocyte colony stimulatingfactor (G-CSF), fibroblast growth factor (FGF), nerve growth factor(NFG), neurotrophins, epidermal growth factor (EGF), platelet derivedgrowth factor (PDGF), insulin-like growth factor (IGF), hepatocytegrowth factor (HGF), erythropoietin (EPO), thrombopoietin 9TPO),myostatin (GDF-8), growth differentiation factor-9 (GDF-9), acidicfibroblast growth factor (aFGF or FGF-1), or basic fibroblast growthfactor (bFGF or FGF-2).

Suitable bioagents useful in accordance with the invention include, butare not limited to, DNA molecules, RNA molecules, antisense nucleicacids, ribozymes, plasmids, expression vectors, marker proteins,transcription or elongation factors, cell cycle control proteins,kinases, phosphatases, DNA repair proteins, oncogenes, tumorsuppressors, angiogenic proteins, anti-angiogenic proteins, cell surfacereceptors, accessory signaling molecules, transport proteins, enzymes,anti-bacterial agents, anti-viral agents, antigens, immunogens,apoptosis-inducing agents, anti-apoptosis agents, cytotoxins, andantibodies against immunosuppression (e.g., transforming growth factor(TGF)-beta antibody or antagonists, and adenosine (A2aR) antagonists).

Bioagents suitable for use in the present invention may also include,but are not limited to, growth factors, hormones, neurotransmitters,neurotransmitter or growth factor receptors, interferons, interleukins,chemokines, cytokines, colony stimulating factors, chemotactic factors,MMP-sensitive substrate, extracellular matrix components. Exemplarybioagents include, but are not limited to, growth hormone, parathyroidhormone (PTH), bone morphogenetic protein (BMP), transforming growthfactor-α (TGF-α), TGF-β, fibroblast growth factor (FGF),granulocyte/macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), nerve growth factor (NFG),neurotrophins, epidermal growth factor (EGF), platelet derived growthfactor (PDGF), insulin-like growth factor (IGF), vascular endothelialcell growth factor (VEGF), hepatocyte growth factor (HGF),erythropoietin (EPO), thrombopoietin 9TPO), myostatin (GDF-8), growthdifferentiation factor-9 (GDF-9), acidic fibroblast growth factor (aFGFor FGF-1), basic fibroblast growth factor (bFGF or FGF-2), interferon-γ(γ-IFN), IFN-α, tumor necrosis factor (TNF), fibrin, collagen,fibronectin, vitronectin, hyaluronic acid, RGD-containing peptides orpolypeptides, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10,IL-12, IL-15, IL-17, IL-18, FLT-3 ligand, and CD40 ligand. Splicevariants of any of the above mentioned proteins, and small moleculeagonists or antagonists thereof that may be used advantageously to altera function of a cell are also contemplated herein.

In some embodiments, the bioagent comprises one or more adjuvants.Adjuvants are compounds that enhance the specific immune responseagainst antigens. Adjuvants are added to vaccines to stimulate theimmune system's response to the target antigen. Adjuvants may enhancethe immunogenicity of highly purified or recombinant antigens. Adjuvantsmay reduce the amount of antigen or the number of immunizations need toprotective immunity. For example, adjuvants may activateantibody-secreting B cells to produce a higher amount of antibodies.Alternatively, adjuvants can act as a depot for an antigen, present theantigen over a longer period of time, which could help maximize theimmune response and provide a longer-lasting protection. Adjuvants mayalso be used to enhance the efficacy of a vaccine by helping to modifythe immune response to particular types of immune system cells, forexample, by activating T cells instead of antibody-secreting B cellsdepending on the purpose of the vaccine. Adjuvants are also used in theproduction of antibodies from immunized animals (Petrovskyl et al, 2002,Immunology and Cell Biology 82: 488-496).

Adjuvants can be classified according to their source, mechanism ofaction or physicochemical properties. For example, adjuvants can beclassified into three groups: (i) active immunostimulants, beingsubstances that increase the immune response to the antigen; (ii)carriers, being immunogenic proteins that provide T-cell help; and (iii)vehicle adjuvants, being oil emulsions or liposomes that serve as amatrix for antigens as well as stimulating the immune response (EdelmanR. 1992, AIDS Res. Hum. Retroviruses 8: 1409-11). An alternativeadjuvant classification divides adjuvants according to administrationroute, namely mucosal or parenteral. A third classification dividesadjuvants into alum salts and other mineral adjuvants; tensoactiveagents; bacterial derivatives; vehicles and slow release materials orcytokines (Byars et al., 1990, Laboratory Methods in Immunology: 39-51).A fourth classification divides adjuvants into the following groups:gel-based adjuvants, tensoactive agents, bacterial products, oilemulsions, particulated adjuvants, fusion proteins or lipopeptides(Jennings R et al., 1998, Dev. Biol. Stand, 92: 19-28).

The vaccine compositions of the present invention may comprise one ormore adjuvants. Adjuvants suitable for use in the present inventioninclude, but are not limited to, mineral salt-based adjuvants such asalum-based adjuvants, calcium-based adjuvants, iron-based adjuvants,zirconium-based adjuvants; particulate adjuvants; mucosal adjuvants;tensoactive adjuvants; bacteria-derived adjuvants; oil-based adjuvants;cytokines, liposome adjuvants, polymeric microsphere adjuvants,carbohydrate adjuvants.

Exemplary adjuvants include, but are not limited to, aluminiumhydroxide, aluminum phosphate, calcium phosphate, Quil A, Quil A derivedsaponin QS-21, or other types of saponins, Detox, ISCOMs, cell wallpeptidoglycan or lipopolysaccharide of Gram-negative bacteria, trehalosedimycolate, bacterial nucleic acids such as DNA containing CpG motifs,FIA, Montanide, Adjuvant 65, Freund's complete adjuvant, Freund'sincomplete adjuvant, Lipovant, interferon, granulocyte-macrophage colonystimulating factor (GM-CSF), AS03, AS04, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, STING, Toll-likereceptor ligand, CD40L, ovalbumin (OVA), monophosphoryl lipid A (MPL),poly(I:C), a combination of LPS (or MPLA) and OxPAPC, MF59, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), poly (DL-lactide-coglycolide)microspheres, paraffin oil, squalene, virosome, gamma inulin, glucans,dextrans, lentinans, glucomannans and galactomannans,pathogen-associated molecular patterns (PAMPs), damage-associatedmolecular pattern molecules (DAMPs), antibodies against immunesuppressive molecules (e.g., antibody or antagonist against transforminggrowth factor (TGF)-beta, A2aR antagonists), Freund's complete adjuvant,Freund's incomplete adjuvant, lipopolysaccharides (LPS), Fas ligand,Trail, lymphotactin, Mannan (M-FP), APG-2, Hsp70 and Hsp90.

Adjuvants may comprise any number of delivery systems, for example,mineral salts, surface active agents, synthetic micro particles,oil-in-water emulsions, immunostimulatory complexes, liposomes,virosomes, and virus-like particles. Adjuvants may further comprise oneor more potentiators of the immune response such as microbialderivatives (e.g., bacterial products, toxins such as cholera toxin andheat labile toxin from E. coli, lipids, lipoproteins, nucleic acids,peptidogylcans, carbohydrates, peptides), cells, cytokines, (e.g.,dendritic cells, IL-12, and GM-CSF), hormones, and small molecules.Adjuvants suitable for use in the present invention include, but are notlimited to, oil-based adjuvants (e.g., Freund's adjuvant), CpGoligonucleotides, aluminum salt adjuvants, calcium salt adjuvants,emulsions and surfactant-based formulations (e.g., MF59, AS02,montanide, ISA-51, ISA-720, and QA21). For a review of improvements invaccine adjuvants, see Pashine et al. 2005, Nature Med. 11(4): S63-S68.

In one embodiment, the adjuvant comprises or consists of one or moretoll-like receptor (TLR) agonists. In one embodiment, the TLR agonist isa pathogen associated agonist selected from the group consisting oftriacylated lipopeptides (gram positive bacteria), Peptidoglycan (grampositive bacteria), bacterial lipoprotein, lipoteichoic acid, LPS(Porphyromonas gingivalis, Leptospira interrogans), GPI-anchor proteins(Trypanosoma cruzi), neisserial porins, hemagglutinin (MV),phospholipomannan (Candida), LAM (Mycobacteria), ssRNA virus (WNV),dsRNA virus (RSV, MCMV), LPS (Gram-negative bacteria), F-protein (RSV),mannan (Candida), glycoinositolphospholipids (Trypanosoma), envelopeproteins (RSV and MMTV), flagellin (Flagellated bacteria),phenol-soluble modulin (Staphylococcus epidermidis), diacylatedlipopeptides (Mycoplasma), LTA (Streptococcus), zymosan (Saccharomyces),viral ssRNA (Influenza, VSV, HIV, HCV), ssRNA from RNA virus, dsDNAviruses (HSV, MCMV), hemozoin (Plasmodium), and unmethylated CpG DNA(bacteria and viruses). In one embodiment, the TLR agonist is asynthetic ligand selected from the group consisting of Pam3Cys, CFA,MALP2, Pam2Cys, FSL-1, Hib-OMPC, Poly I:C; poly A:U, AGP, MPL A, RC-529,MDF2P, CFA, fiagellin, MALP-2, Pam2Cys, FSL-1, Guanosine analogs,imidazoquinolines (e.g. Imiquimod, Aldara® R848, Esiquimod®),loxoribine, imidazoquinolines, Loxoribine, ssPolyU, 3M-012, andCpG-oligonucleotides.

In some embodiments, the adjuvant comprises granulocyte-macrophagecolony-stimulating factor (GM-CSF). Granulocyte-macrophagecolony-stimulating factor (GM-CSF) is a protein secreted by macrophages,T cells, mast cells, endothelial cells and fibroblasts. Specifically,GM-CSF is a cytokine that functions as a white blood cell growth factor.GM-CSF stimulates stem cells to produce granulocytes and monocytes.Monocytes exit the blood stream, migrate into tissue, and subsequentlymature into macrophages. GM-CSF also has the ability to recruit andprogram antigen-presenting cells.

GM-CSF polypeptides can be isolated from endogenous sources orsynthesized in vivo or in vitro. Endogenous GM-CSF polypeptides areisolated from healthy human tissue. Synthetic GM-CSF polypeptides aresynthesized following transfection or transformation of template DNAinto a host organism or cell, e.g., a mammal or cultured human cellline. Alternatively, synthetic GM-CSF polypeptides are synthesized bypolymerase chain reaction (PCR) or other well-known methods in the art(Sambrook, J., et al., Molecular Cloning: A Laboratory Manual. ColdSpring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), the entirecontents of the foregoing publication is incorporated herein byreference).

GM-CSF polypeptides are modified to increase protein stability in vivo.Alternatively, GM-CSF polypeptides are engineered to be more or lessimmunogenic. GM-CSF polypeptides are recombinant. Alternatively, GM-CSFpolypeptides are humanized derivatives of mammalian GM-CSF polypeptides.Exemplary mammalian species from which GM-CSF polypeptides are derivedinclude, but are not limited to, mouse, rat, hamster, guinea pig, goat,bird, cat, dog, monkey, or primate. In some embodiments, GM-CSF is arecombinant human protein (PeproTech, Catalog #300-03). In otherembodiments, GM-CSF is a recombinant murine (mouse) protein (PeproTech,Catalog #315-03). In yet another embodiment, GM-CSF is a humanizedderivative of a recombinant mouse protein.

In some embodiments, the adjuvant comprises cytosine-Guanosine (CpG)Oligonucleotide (CpG-ODN) sequences, e.g., the adjuvant comprises CpGdinucleotides or CpG oligonucleotides. CpG sites are regions ofdeoxyribonucleic acid where a cysteine nucleotide occurs next to aguanine nucleotide in the linear sequence of bases along its length (the“p” represents the phosphate linkage between them and distinguishes themfrom a cytosine-guanine complementary base pairing). CpG sites play apivotal role in DNA methylation, which is one of several endogenousmechanisms cells use to silence gene expression. Methylation of CpGsites within promoter elements can lead to gene silencing. CpG-ODNsequences found in bacterial DNA are potent immunomodulators thatstimulate activation of an antigen presenting cell, for example,dendritic cell, leading to specific T-cell responses.

CpG oligonucleotides for use in the vaccine compositions of the presentinvention can be isolated from endogenous sources or synthesized in vivoor in vitro. Exemplary sources of endogenous CpG oligonucleotidesinclude, but are not limited to, microorganisms, bacteria, fungi,protozoa, viruses, molds, or parasites. Synthetic CpG oligonucleotidesare synthesized following transfection or transformation of template DNAinto a host organism. Alternatively, Synthetic CpG oligonucleotides aresynthesized by polymerase chain reaction (PCR) or other well-knownmethods in the art.

CpG oligonucleotides are presented for cellular uptake by dendriticcells. In one embodiment, naked CpG oligonucleotides are used in thevaccine compositions of the invention. The term “naked” is used todescribe an isolated endogenous or synthetic polynucleotide (oroligonucleotide) that is free of additional substituents. In anotherembodiment, CpG oligonucleotides are bound to one or more compounds toincrease the efficiency of cellular uptake. Alternatively, or inaddition, CpG oligonucleotides are bound to one or more compounds toincrease the stability of the oligonucleotide within the vaccinecomposition and/or dendritic cells.

CpG oligonucleotides are condensed prior to cellular uptake. In someembodiments, CpG oligonucleotides are condensed using polyethylimine(PEI), a cationic polymer that increases the efficiency of cellularuptake into dendritic cells. The amine-rick polycation, PEI, condensesplasmid DNA via association with DNA phosphate groups, resulting insmall, positively charge condensates facilitating cell membraneassociation and DNA uptake into cells (Godbey et al., 1999, Proc NatlAcad Sci USA, 96(9): 5177-81).

In some embodiments, the bioagents, e.g., adjuvants, are linked to thesolid substrate, e.g.,beads. Bioagents can be conjugated to the surfaceof the solid substrate using any of a variety of methods known to thoseof skill in the art for conjugating proteins or peptides with othermolecules. For example, Hermanson, Bioconjugate Techniques (2nd Ed.,Academic Press (2008)) and Niemeyr, Bioconjugation Protocols: Strategies& Methods, in Methods In Molecular Biology (Humana Press, 2004), providea number of methods and techniques for conjugating peptides to othermolecules. The bioagents can be coupled or conjugated to the surface ofthe solid substrate covalently or non-covalently. The covalent linkagebetween the bioagents and the surface can also be mediated by a linker.The non-covalent linkage between the bioagents and the surface can bebased on ionic interactions, van der Waals interactions, dipole-dipoleinteractions, hydrogen bonds, electrostatic interactions, and/or shaperecognition interactions.

The bioagents in the vaccine compositions of the present invention arecapable of recruiting and/or activating an immune cell in a subject. Animmune cell refers to any cell of the immune system that functions toprotect the body against both infectious disease and foreign invaders.Exemplary immune cells include, but are not limited to, a T cell, a Bcell, an antigen-presenting cell, a dendritic cell, a macrophage, agranulocytes, a monocyte, a neutrophil, and a natural killer (NK) cell.

In some embodiments, the immune cell is an antigen-presenting cell.Antigen-presenting cells (APCs), also known as accessory cells, arecells that display antigen complexed with major histocompatibilitycomplexes (MHCs) on their surfaces. Antigen-presenting cells processantigens and present them to the T cells of the immune system.Antigen-presenting cells can be found in a variety of tissue types.Classical antigen-presenting cells, including macrophages, B cells, anddendritic cells, associate with an MHC class II molecule to presentforeign antigens to helper T cells. While other cell types, e.g., mastcells, basophils, eosinophils, and group 3 innate lymphoid cells(ILC3s), that can express MHC class II molecules, may also serve asantigen-presenting cells (Kambayashi and Laufer, 2014, Nature ReviewsImmunology 14, 719-730). Antigen-presenting cells may also include anycells, e.g., nucleated cells, that are capable of displaying endogenouspeptides on the cell surface to cytotoxic T cells, typically using anMHC class I molecule. In addition to the MHC family of proteins, antigenpresentation relies on other specialized signaling molecules on thesurfaces of both APCs and T cells.

Antigen-presenting cells are very efficient at internalizing antigens,either by phagocytosis (macrophages and dendritic cells) or byreceptor-mediated endocytosis (B cells), processing the antigen intopeptide fragments and then displaying those peptides, bound to a classII MHC molecule, on their membrane. T cells recognize and interact withthe antigen-class II MHC molecule complex on the membrane of theantigen-presenting cell. An additional co-stimulatory signal is thenproduced by the antigen-presenting cell, leading to activation of the Tcell. The expression of co-stimulatory molecules and MHC class II aredefining features of professional APCs.

Antigen-presenting cells (APCs) are vital for effective adaptive immuneresponse because the functioning of both cytotoxic and helper T cells isdependent on APCs. Antigen presentation allows for specificity ofadaptive immunity and can contribute to immune responses against bothintracellular and extracellular pathogens.

The vaccine compositions of the present invention may further comprise ascaffold comprising a biomaterial and capable of recruiting andactivating an immune cell in a subject. Thus, the present invention alsoprovides vaccine compositions comprising a scaffold comprising abiomaterial and capable of recruiting and activating an immune cell in asubject; and an opsonin-bound or lectin-bound pathogen construct. Thescaffold provides a physical structure upon which or into which theopsonin-bound or lectin-bound pathogen construct or the bioagent canassociate or attach. The opsonin-bound or lectin-bound pathogenconstruct can be encapsulated within the scaffold for delivery oradministration to a subject, therefore preventing any undesired leakageof pathogen from the vaccine composition. In some embodiments, thevaccine compositions further comprise one or more bioagents as describedherein. The bioagent is capable of recruiting and/or activating animmune cell in a subject. In some embodiments, the vaccine compositionscomprise granulocyte macrophage colony stimulating factor (GM-CSF). Inother embodiments, the vaccine compositions comprise acytosine-guanosine oligonucleotide (CpG-ODN) sequence. In yet anotherembodiment, the vaccine compositions comprise the combination ofgranulocyte macrophage colony stimulating factor (GM-CSF) andcytosine-guanosine oligonucleotide (CpG-ODN) sequence.

The scaffolds comprise a biocompatible material that is not toxic orimmunogenic. As used herein, the term “biocompatible material” refers toany material that does not deteriorate appreciably and does not induce asignificant immune response or deleterious tissue reaction, e.g., toxicreaction or significant irritation, over time when implanted into orplaced adjacent to the biological tissue of a subject, or that does notinduce blood clotting or coagulation when it comes in contact withblood.

The scaffolds can be biodegradable in the body. In some embodiments, thescaffold composition degrades at a predetermined rate based on aphysical parameter selected from the group consisting of temperature,pH, hydration status, and porosity, the cross-link density, type, andchemistry or the susceptibility of main chain linkages to degradation orit degrades at a predetermined rate based on a ratio of chemicalpolymers. For example, a high molecular weight polymer comprised ofsolely lactide degrades over a period of years, e.g., 1-2 years, while alow molecular weight polymer comprised of a 50:50 mixture of lactide andglycolide degrades in a matter of weeks, e.g., 1, 2, 3, 4, 6, 10 weeks.A calcium cross-linked gels composed of high molecular weight, highguluronic acid alginate degrade over several months (1, 2, 4, 6, 8, 10,12 months) to years (1, 2, 5 years) in vivo, while a gel comprised oflow molecular weight alginate, and/or alginate that has been partiallyoxidized, will degrade in a matter of weeks.

Exemplary biomaterals suitable for use as scaffolds in the presentinvention include glycosaminoglycan, silk, fibrin, MATRIGEL®,poly-ethyleneglycol (PEG), polyhydroxy ethyl methacrylate,polyacrylamide, poly (N-vinyl pyrolidone), (PGA), polylactic-co-glycolic acid (PLGA), poly e-carpolactone (PCL), polyethyleneoxide, poly propylene fumarate (PPF), poly acrylic acid (PAA),polyhydroxybutyric acid, hydrolysed polyacrylonitrile, polymethacrylicacid, polyethylene amine, esters of alginic acid; pectinic acid; andalginate, fully or partially oxidized alginate, hyaluronic acid, carboxymethyl cellulose, heparin, heparin sulfate, chitosan, carboxymethylchitosan, chitin, pullulan, gellan, xanthan, collagen, gelatin,carboxymethyl starch, carboxymethyl dextran, chondroitin sulfate,cationic guar, cationic starch, and combinations thereof. In certainembodiments, the biomaterial is selected from the group consisting ofpoly(L-lactide-co-glycolide) acid (PLGA), mesoporous silica, cryogel,and combinations thereof.

In some embodiments, the scaffold comprises a macroporouspoly-lactide-co-glycolide (PLG). Vaccine compositions comprising PLGscaffolds are capable of releasing the encapsulated composition, e.g.,an opsonin-bound or lectin-bound pathogen construct, or a bioagent, fromthe scaffolds, gradually over the period of time, for example, over thenext days or weeks, after administration to the target site in or on thesubject. The rate of releasing the encapsulated pathogen from thescaffolds can therefore by regulated in a temporal manner. As a result,the immune responses elicited by the pathogen in the vaccinecompositions can also be controlled temporally.

In some embodiments, the scaffolds comprise mesoporous silica (MPS). Inother embodiments, the scaffolds comprise cryogels. See, e.g.,International Patent Publication No. WO 2012/149358A1. The entirecontents of the foregoing patent application are incorporated herein byreference.

In some embodiments, the scaffolds comprise a non-biodegradable materialor are resistant to breakdown in the body. Relatively permanent(degradation resistant) scaffold compositions include metals and somepolymers such as silk and plastic.

In some embodiments, the scaffolds, e.g., hydrogels, comprisebiomaterials that are modified, e.g., sites on the biomaterial aremodified with a functional group, e.g., a methacrylic acid group(methacrylate (MA)) or an acrylic acid group (acrylate). The degree ofmethacrylation can be varied from 1% to 90%. Above 90%, the chemicalmodification may reduce solubility of the polymer water-solubility.Exemplary modified hydrogels are MA-alginate (methacrylated alginate) orMA-gelatin. In the case of MA-alginate or MA-gelatin, 50% corresponds tothe degree of methacrylation of alginate or gelatin. This means thatevery other repeat unit contains a methacrylated group.

Biomaterials for use in the scaffolds of the present invention can alsobe modified with acrylated groups instead of methacrylated groups. Theproduct would then be referred to as an acrylated-polymer. The degree ofmethacrylation (or acrylation) can be varied for most polymers. Further,alginate or gelatin may be modified with click moieties to produceclick-alginate or click-gelatin cryogels (see, e.g., InternationalPatent Application Publication No. WO2015154078, the entire contents ofthe foregoing patent application are incorporated herein by reference),In addition, click alginate may be oxidized and further proceeded usingeither ammonia borane or sodium chlorite to elimination aldehydes priorto click conjugation to produce a biodegradable click alginate cryogel(see, e.g., International Patent Application No. PCT/US16/058866, theentire contents of the foregoing patent application are incorporatedherein by reference),

The scaffolds of the present invention may comprise an external surface.Alternatively, or in addition, the scaffolds may comprise an internalsurface. External or internal surfaces of the scaffolds of the presentinvention may be solid or porous. Pore size of the scaffolds can be lessthan about 10 nm, between about 100 nm-20 μm, or greater than about 20μm, e.g., up to and including 1000 μm in diameter. For example, thepores may be nanoporous, microporous, or macroporous. For example, thediameter of nanopores are less than about 10 nm; micropore are in therange of about 100 μm-20 μm in diameter; and, macropores are greaterthan about 20 μm, e.g., greater than about 100 μm, e.g., greater thanabout 400 μm, e.g., greater than 600 μm or greater than 800 μm.

In some embodiments, the scaffolds of the present invention areorganized in a variety of geometric shapes (e.g., discs, beads,pellets), niches, planar layers (e.g., thin sheets). For example, discsof about 0.1-200 millimeters in diameter, e.g., 5, 10, 20, 40, 50millimeters may be implanted subcutaneously. The disc may have athickness of 0.1 to 10 millimeters, e.g., 1, 2, 5 millimeters. The discsare readily compressed or lyophilized for administration to a patient.An exemplary disc for subcutaneous administration has the followingdimensions: 8 millimeters in diameter and 1 millimeter in thickness. Insome embodiments, the scaffolds may comprise multiple components.Multicomponent scaffolds are optionally constructed in concentric layerseach of which is characterized by different physical qualities such asthe percentage of polymer, the percentage of cros slinking of polymer,chemical composition of the hydrogel, pore size, porosity, and porearchitecture, stiffness, toughness, ductility, viscoelasticity, and/orpharmaceutical composition.

In some embodiments, the scaffold further comprises one or morebioagents as described herein. In some embodiments, the bioagent iscovalently linked to the scaffold or the solid structure, e.g., beads,within the scaffold, keeping the bioagent relatively immobilized in oron the scaffold or the solid structure, e.g., beads, within thescaffold. In other embodiments, the bioagent is non-covalentlyassociated with the scaffold. Non-covalent bonds include electrostatic,hydrogen, vander Waals, and hydrophobic interaction. In someembodiments, the scaffold comprises granulocyte macrophage colonystimulating factor (GM-CSF). In other embodiments, the scaffoldcomprises cytosine-guanosine oligonucleotide (CpG-ODN) sequences. Incertain embodiments, the scaffold comprises both GM-CSF and CpG-ODN.

The bioagents are added to the scaffold using known methods in the artincluding surface absorption, physical immobilization, e.g., using aphase change to entrap the substance in the scaffold material. Forexample, a bioagent, e.g., a growth factor, is mixed with the scaffoldwhile it is in an aqueous or liquid phase, and after a change inenvironmental conditions (e.g., pH, temperature, ion concentration), theliquid gels or solidifies thereby entrapping the bioagent.Alternatively, covalent coupling, e.g., using alkylating or acylatingagents, is used to provide a stable, long term presentation of abioagent on the scaffold in a defined conformation. Exemplary reagentsfor covalent coupling of such substances are provided in the tablebelow. Approaches to coupling of bioagents, e.g., proteins or peptides,to scaffold polymers are discussed in Hirano and Mooney, 2005, AdvancedMaterials, 16(1): 17-25. Other useful bonding chemistries also includethose discussed in Hermanson, 1996, Bioconjugate Techniques, 152-185.

Methods to covalently couple peptides/proteins to polymers FunctionalGroup Reacting groups on of Polymer Coupling reagents and cross-linkerproteins/peptides —OH Cyanogen bromide (CNBr) —NH₂ Cyanuric chloride4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl- morpholinium chloride(DMT-MM) —NH₂ Diisocyanate compounds —NH₂ Diisothoncyanate compounds —OHGlutaraldehyde Succinic anhydride —NH₂ Nitrous Acid —NH₂ Hydrazine +nitrous acid —SH —Ph—OH —NH₂ Carbodiimide compounds (e.g., EDC, DCC)[a]—COOH DMT-MM Azide Copper catalyst -alkyne Azide None -DBCO orCycooctyne Tetrazine None -TCO —NH₂ Sortase enzyme Peptide —O—NH₂Pyridoxamine N-terminus —COOH Thionyl chloride —NH₂ N-hydroxysuccinimideN-hydroxysulfosuccinimide + EDC —SH Disulfide compound —SH MaleimideNone —SH iodoacetate None SH [a]EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; DCC: dicyclohexylcarbodiimide

The scaffolds are assembled using methods known in the art, e.g.,injection molding, lyophilization of preformed structures, printing,self-assembly, phase inversion, solvent casting, melt processing, gasfoaming, fiber forming/processing, particulate leaching or a combinationthereof. The assembled scaffolds are then used to prepare the vaccinecompositions of the present invention.

In some embodiments, the scaffolds are suitable for implantation. Forexample, the scaffolds are prepared using poly-lactide-co-glycolide(PLG). The vaccine compositions comprising the PLG scaffolds, ifimplanted, can be easily removed from the subject after vaccination. Forexample, in the case where too much immune response or undesired sideeffects are initiated after vaccination, the implanted vaccinecompositions can be removed from the subject. In some embodiments, thescaffolds are suitable for injection. For example, the scaffolds arecreated outside of the body as macroporous scaffolds. The scaffold canbe injected into the body because the pores collapse and the gel becomesvery small and can fit through a needle. See, e.g., WO 12/149358; andBencherif et al. Proc. Natl. Acad. Sci. USA 109.48(2012):19590-5. Theentire contents of each of the foregoing references are incorporatedherein by reference.

The vaccine compositions of the present invention may comprise one typeof pathogen, or they may comprise multiple types of pathogens, e.g., atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35,40, 45, 50 or more types of pathogens. The vaccine compositions may alsocontain one or more types of adjuvants that program the immune cells torecognize antigen and enhance antigen presentation as described herein.

The vaccine compositions of the present invention may be systemicallyadministered, e.g., enterally administered (e.g., orally, buccally,sublingually, or rectally) or parenterally administered (e.g.,intravenously, intra-arterially, intraosseously, intra-muscularly,intracerebrally, intracerebroventricularly, intrathecally, orsubcutaneously). Other suitable modes of administration of the vaccinecompositions of the invention include hypodermal, intraperitoneal,intraocular, intranasal, transdermal (e.g., via a skin patch), epidural,intracranial, percutaneous, intravaginal, intrauterineal, intravitreal,or transmucosal administration, or administration via injection, or viaimplantation.

As shown in the Examples, in accordance to the vaccine compositionsdescribed herein, subcutaneous implantation or injection of the vaccinecompositions in mice successfully protected the mice from a lethal doseof bacterial infection. Specifically, mice immunized with the vaccinecompositions of the present invention exhibited a significantlyprolonged survival time and a significantly reduced titer of pathogen invarious organs when compared with the control mice. Accordingly, in someembodiments, the vaccine composition is suitable for implantation, e.g.,subcutaneous implantation. In some embodiments, the vaccine compositionis suitable for injection in a subject. In certain embodiments, thevaccine composition is suitable for oral administration in a subject.For example, the vaccine composition can be in the form of a pill, atablet, a capsule, a soft gel, a chewable, a powder, an emulsion, or anaqueous solution for oral administration.

The vaccine compositions of the present invention possess a distinctadvantage over existing vaccines in that only one single administrationis sufficient to elicit a sustained immune response, e.g., aantibody-mediated and/or cell-mediated immune response, against targetedpathogens. Indeed, as shown in Example 1, a single dose of the vaccinecomposition of the invention can protect the vaccinated mice frombacteria challenge over a period of at least 90 days.

In addition, the vaccine compostions confer another significantadvantage over available vaccines in the art in that they can be easilyremoved from a subject after implantation. For example, if too muchimmune response or undesired side effects are initiated after thesubject receives the vaccine compositions, the vaccine compositions,e.g., PLG scaffold vaccine compositoons, can be readily removed from thesubject. In contract, current existing vaccines cannot be removed oncethey are injected in the subjects.

The vaccine compositions of the present invention are highly stable andcapable of being portable and being stored at room temperature withoutthe need for refrigeration. Upon lyophilization, the vaccinecompositions may have a shelf life of about 1 day to about 1 year, e.g.,about 10 days to about 1 year, about 30 days, to about 1 year, about 2months to about 1 year, about 3 months to about 1 year, about 4 monthsto about 1 year, about 5 months to about 1 year, about 6 months to about1 year, about 7 months to about 1 year, about 8 months to about 1 year,about 9 months to about 1 year, about 10 months to about 1 year. In someembodiments, the vaccine composition has a shelf life of at least 1year, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

The vaccine compositions of the present invention allow the rapid anddirect isolation of pathogens circulating in a blood sample from apatient with infectious disease including both known and unknownpathogens, pathogens present within other biological fluids, orpathogens present in an in vitro culture, and hence greatly increase theimmune response as compared to conventional antibiotic therapies. Theclaimed vaccine compositions can also be used against pathogens that aredifficult to isolate and purify. The vaccine compositions of the presentinvention also possess additional improvements over existing vaccines.For example, vaccination using the claimed vaccine compositions canoccur in a more controlled, localized and safer manner, withoutcompromising the efficacy of the vaccine compositions. The improvedstability of the claimed vaccines allows them to be portable and to beused for long term storage at room temperature without the need ofrefrigeration. Furthermore, the vaccine compositions can be multivalentvaccines when more than one type of pathogens are included in thecompositions, and can also be used to vaccinate against differentspecies or strains of a given pathogen. These improvements circumventthe major limitations of current pathogen vaccines, and would be ofgreat interest to the public, especially during the time of an epidemic,for example, for populations in developing countries, or of great valuefor military uses, where vaccines that are readily available are highlydesired. For example, pathogens can be isolated directly and rapidlyusing an engineered lectin, e.g., the engineered MBL, from a subject,e.g., a human, having or at risk of having an infectious disease,neutralized by antibiotics treatment, incorporated into a scaffoldcomposition, e.g., a PLG scaffold, and then administered back to thesame human subject, or a different human subject, for treatinginfectious disease. Alternatively, the vaccine composition can belyophilized and stored at room temperature for about 1 day to about 1year, and then administered to a subject to induce an immune responseagainst the pathogen or pathogen fragment within the vaccinecompositions, thereby treating the infectious disease. This is extremelybeneficial especially in the case where ring vaccination is highlydesired to treat infectious diseases or prevent the spread of infectiouspathogens. The vaccine compositions of the present invention can be madereadily available and then administered to infected individuals or anyother individuals that come in contact with the infected group or are atrisk of being infected.

The present invention also provides stable scaffold compositions. Thestable scaffold compositions comprise a biomaterial and capable ofrecruiting and activating an immune cell in a subject, wherein thescaffold is lyophilized, and wherein the scaffold has a shelf life ofabout 30 days to about 1 year.

In another aspect, the present invention provides stable opsonin-boundor lectin-bound pathogen constructs. The opsonin-bound or lectin-boundpathogen constructs comprise a pathogen or fragment thereof derived froma subject bound to an opsonin or a lectin, wherein the opsonin-bound orlectin-bound pathogen construct is lyophilized, and wherein theopsonin-bound or lectin-bound pathogen construct has a shelf life ofabout 30 days to about 1 year.

In yet another aspect, the prevent invention provides scaffoldcompositions. The scaffold compositions comprise a biomaterial and arecapable of recruiting and activating an immune cell in a subject,wherein the scaffold comprises a solid substrate, e.g., beads, e.g.,magnetic beads, and wherein the solid substrate is suitable forattachment of a pathogen. The scaffold compositions of the invention arestable and can be readily available, for example, during a war or duringa pandemic. Any pathogen or pathogen fragment that is capable ofinducing an infectious disease in a subject, as described herein, may beintroduced in the scaffold compositions of the invention in order togenerate vaccine compositions. Pathogens or pathogen fragments can beintroduced into the scaffold compositions of the invention once they areisolated, for example, from a sample of a subject, e.g., a human, orfrom an in vitro culture, then incorporated into the scaffoldcomposition, and then administered to a subject for treating infectiousdisease. These scaffold compositions are very useful and highlydesirable, for example, during the time of an epidemic, or of greatvalue for military uses, where vaccines that are readily available arehighly desired.

III. Formulations

The vaccine compositions of the present invention can be used directlyfor purposes of treatment and prophylaxis of various diseases, e.g.,infectious diseases. The vaccine compositions of the invention can beformulated using one or more physiologically acceptable carriers orexcipients. For example, where a composition is formulated as a liquid,it may comprise sterile saline, a dextrose solution, or a bufferedsolution, or other pharmaceutically acceptable sterile fluid. In someembodiments, the formulations are for intradermal or subcutaneousadministration. In some embodiments, the formulations are for inhalationor insufflation (either through the mouth or the nose). In someembodiments, the formulations are for oral, buccal, parenteral, vaginal,or rectal administration. The term parenteral as used herein includessubcutaneous, intracutaneous, intravenous, intramuscular,intra-articular, intraarterial, intrasynovial, intrasternal,intrathecal, intralesional and intracranial injection or infusiontechniques. In some embodiments, the formulations are for implantation.

Preferably, the vaccine compositions are formulated to provide increasedchemical stability of the opsonin-bound or lectin-bound pathogenconstruct or the bioagent during storage and transportation. Forexample, in one embodiment, the formulation prevents or reduces proteinoligomerization or aggregation. In another example, the formulationprevents or reduces oxidation of the amino acid residues. Theformulations may be lyophilized or liquid formulations. Lyophilizedformulations have a shelf life of about 1 day to about 1 year, e.g.,about 10 days to about 1 year, about 30 days, to about 1 year, about 2months to about 1 year, about 3 months to about 1 year, about 4 monthsto about 1 year, about 5 months to about 1 year, about 6 months to about1 year, about 7 months to about 1 year, about 8 months to about 1 year,about 9 months to about 1 year, about 10 months to about 1 year. In someembodiments, the formulation has a shelf life of at least 1 year, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

In some embodiments, the vaccine compositions are formulated forinjection. In certain embodiments, the vaccine compositions are sterilelyophilized formulations, substantially free of contaminating cellularmaterial, chemicals, virus, or toxins. In a particular embodiment,formulations for injection are provided in sterile single dosagecontainers. The formulations may or may not contain an addedpreservative. Liquid formulations may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents.

In one embodiment, the formulation comprises liposomes. In oneembodiment, a vaccine composition of the invention is formulated withone or more other therapeutic agents used for the treatment ofinfectious diseases.

The vaccine compositions of the invention may be formulated aspharmaceutical compositions and can include one or more pharmaceuticallyacceptable carriers, additives, or vehicles. In one embodiment, the oneor more pharmaceutically acceptable carriers, additives, or vehicles isselected from the group consisting of ion exchangers, alumina, aluminumstearate, lecithin, self-emulsifying drug delivery systems (SEDDS) suchas d-I-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins may also be advantageously used to enhancedelivery of compounds of the formulae described herein. The vaccinecompositions of the invention may also comprise a pharmaceuticallyacceptable acid, base or buffer to enhance the stability of theformulated compound or its delivery form.

In some embodiments, the vaccine compositions of the invention are inthe form of a solution or powder for inhalation and/or nasaladministration. Such compositions may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives, are useful in the preparation of injectables.These oil solutions or suspensions may also contain a long-chain alcoholdiluent or dispersant, or carboxymethyl cellulose or similar dispersingagents which are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and or suspensions. Othercommonly used surfactants such as Tweens or Spans and/or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

In some embodiments, the vaccine compositions of the invention are inthe form of an orally acceptable dosage form including, but not limitedto, solid drugs such as pills, tablets, granules, soft gels, chewable,powders, and capsules; liquid drugs such as emulsions and aqueoussuspensions, dispersions and solutions; inhalants and lyophilized drugs.In the case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions and/or emulsions are administered orally, the activeingredient may be suspended or dissolved in an oily phase is combinedwith emulsifying and/or suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added. Thesedrugs can be prepared by conventional pharmaceutical practices.

IV. Methods of the Invention

The vaccine compositions of the present invention are useful for theprophylaxis and treatment of various diseases, e.g., infectiousdiseases. As shown in the Examples presented herein, immunization ofmice with vaccine compositions of the present invention, e.g., a vaccinecomprising a lectin-bound pathogen construct with CpG and GM-CSFadjuvants within a PLG or MPS scaffold, successfully protected mice froma lethal dose of bacteria, resulting in a significantly prolongedsurvival time and a reduced titer of pathogen in various organs invaccinated mice when compared to controls.

Accordingly, the present invention, in one aspect, provides methods fortreating a pathogen infection in a subject in need thereof. The methodscomprise administering the vaccine composition as described herein,thereby treating the pathogen infection in the subject.

In another aspect, the present invention provides methods of vaccinatinga subject against a pathogen infection. The methods compriseadministering the vaccine composition as described herein, therebyvaccinating the subject against the pathogen infection.

The present invention also provides methods of treating anantibiotic-resistant bacterial infection in a subject in need thereof.The methods comprise administering the vaccine composition as describedherein, thereby treating the antibiotic-resistant bacterial infection inthe subject. In some embodiments, the vaccine composition is specificfor the antibiotic-resistant bacterium in the subject.

The present invention further provides methods of decreasing the levelof a pathogen in a subject having a pathogen infection. The methodscomprise administering the vaccine composition as described herein,thereby decreasing the level of the pathogen in the subject. In someembodiments, the level of the pathogen is decreased in an organ of thesubject. In some embodiments, the organ is selected from the groupconsisting of a lung, a liver, a kidney, and a spleen.

In another aspect, the present invention provides methods of increasingthe survival rate of a subject having a pathogen infection. The methodscomprise administering the vaccine composition as described herein,thereby increasing the survival rate of the subject.

In one aspect, the present invention provides methods of reducing thelevel of pain associated with a pathogen infection in a subject in needthereof. The methods comprise administering the vaccine composition asdescribed herein, thereby reducing the level of pain associated with thepathogen infection in the subject.

In another aspect, the present invention provides methods of reducingthe level of distress associated with a pathogen infection in a subjectin need thereof. The methods comprise administering the vaccinecomposition as described herein, thereby reducing the level of distressassociated with the pathogen infection in the subject.

In some embodiments, the subject suffers from an infectious disease oris at risk of having an infectious disease. Infectious diseases arecaused by pathogenic microorganisms, such as bacteria, viruses,parasites or fungi. The diseases can be spread directly or indirectly,from one person to another. Infectious disease can also be transmittedfrom animals to humans.

Infectious diseases which can be treated prophylactically ortherapeutically with the vaccine compositions of the present inventioninclude, but are not limited to, tuberculosis, measles, meningococcalmeningitis, Pseudomonas aeruginosa, chikungunya, malaria, plaque,HIV/AIDS, pneumonia, rhinoviral diseases, spring-summermeningoencephalitis (SSME), rubella, poliomyelitis, rabies, hepatitis A,hepatitis B, hepatitis C, hepatitis E, buruli ulcer, Ebola virusdisease, yellow fever, Dengue's disease, trachoma, Chagas disease,influenza, smallpox, avian influenza, cholera, Mediterranean fever,undulant fever, Malta fever, contagious abortion, epizootic abortion.Bang's disease, Salmonella food poisoning, enteric paratyphosis,Bacillary dysentery, Pseudotuberculosis, plague, pestilential fever,Vibrios, Circling disease, Weil's disease, Hemorrhagic jaundice(Leptospira icterohaemorrhagiae), canicola fever (L. canicola), dairyworker fever (L. hardjo), Relapsing fever, tick-borne relapsing fever,spirochetal fever, vagabond fever, famine fever, Lyme arthritis,Bannworth's syndrome, tick-borne meningopolyneuritis, erythema chronicummigrans, Vibriosis, Colibacteriosis, colitoxe ia, white scours, gutedema of swine, enteric paratyphosis, Staphylococcal alimentarytoxicosis, staphylococcal gastroenteritis, Canine Corona Virus (CCV) orcanine parvovirus enteritis, feline infectious peritonitis virus,transmissible gastroenteritis (TGE) virus, Hagerman Redmouth Disease(ERMD), Infectious Hematopoietic necrosis (IHN), porcine Actinobacillus(Haemophilus) pleuropneumonia, Hansen's disease, Streptotrichosis,Mycotic Dermatitis of Sheep, Pseudoglanders, Whitmore's disease,Francis' disease, deer-fly fever, rabbit fever, O'Hara disease,Streptobacillary fever, Haverhill fever, epidemic arthritic erythema,sodoku, Shipping or transport fever, hemorrhagic septicemia, Ornithosis,Parrot Fever, Chlamydiosis, North American blastomycosis, Chicagodisease, Gilchrist's disease, Cat Scratch Fever, BenignLymphoreticulosis, Benign nonbacterial Lymphadenitis, BacillaryAngiomatosis, Bacillary Peliosis Hepatis, Query fever, Balkan influenza,Balkan grippe, abattoir fever, Tick-borne fever, pneumorickettsiosis,American Tick Typhus, Tick-borne Typhus Fever, Vesicular Rickettsiosis,Kew Gardens Spotted Fever, Flea-borne Typhus Fever, Endemic TyphusFever, Urban Typhus, Ringworm, Dermatophytosis, Tinea, Trichophytosis,Microsporosis, Jock Itch, Athlete's Foot, Sporothrix schenckii,dimorphic fungus, Cryptococcosis and histoplasmosis, Benign EpidermalMonkeypox, BEMP, Herpesvirus simiae, Simian B Disease, Type C lethargicencephalitis, Yellow fever, Black Vomit, hantavirus pulmonary syndrome,Korean Hemorrhagic Fever, Nephropathia Epidemica, Epidemic HemorrhagicFever, Hemorrhagic Nephrosonephritis, lymphocytic choriomeningitis,California encephalitis/La cros se encephalitis, African HemorrhagicFever, Green or Vervet Monkey Disease, Hydrophobia, Lyssa, Infectioushepatitis, Epidemic hepatitis, Epidemic jaundice, Rubeola, Morbilli,Swine and Equine Influenza, Fowl Plague, Newcastle disease,Piroplasmosis, toxoplasmosis, African Sleeping Sickness, GambianTrypanosomiasis, Rhodesian Trypanosomiasis, Chagas's Disease,Chagas-Mazza Disease, South American Trypanosomiasis, Entamoebahistolytica, Balantidial dysentery, cryptosporidiosis, giardiasis,Microsporidiosis, Anisakiasis, Trichinosis, Angiostrongylosis,eosinophilic meningitis or meningoencephalitis (A. cantonensis),abdominal angiostrongylosis (A. costaricensis), Uncinariasis,Necatoriasis, Hookworm Disease, Capillariasis, Brugiasis, Toxocariasis,Oesophagostomiasis, Strongyloidiasis, Trichostrongylosis, Ascaridiasis,Diphyllobothriasis, Sparganosis, Hydatidosis, Hydatid Disease,Echinococcus granulosis, Cystic hydatid disease, Tapeworm Infection,Schistosoma and the like.

Malignant diseases caused by infectious pathogens may also be treatedusing the vaccine compositions of the invention. Examples of suchdiseases include, but are not limited to, Burkitt lymphoma caused byEBV, Rous sarcoma caused by Rous retrovirus, Kaposi' sarcoma caused byherpes virus type 8, adult T-cell leukemia caused by HTLV-I retrovirus,or hairy cell leukemia caused by HTLV-JJ, and many other tumors andleukemias caused by infectious agents and viruses.

In addition, diseases which are caused by an unknown pathogen,especially malignant or immunological diseases may be treated orprevented using the vaccine compositions of the invention. Non-limitingexamples of these diseases comprise leukemias like acute leukemia, acutelymphocytic leukemia, acute myelocytic leukemias like myeloblastic,promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronicleukemia like chronic myelocytic or granulocytic leukemia, chroniclymphocytic leukemia, polycythemia vera, Sezary cell leukemia, lymphoma,Hodgkin's disease, non-Hodgkin's disease, multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors likesarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, Kaposi's sarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterinecancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, mycosis fungoides, or pagetoidreticulosis.

In some embodiments, the infectious diseases treatable by the methods ofthe present invention are chronic infectious diseases. In someembodiments, the infectious diseases treatable by the methods of thepresent invention are acute infectious diseases.

The methods of the present invention may include administering thevaccine compositions separately or as part of a therapeutic regimen orcombination therapy. The terms “administer,” “administering,” or“administration,” as used herein refer to implanting, absorbing,ingesting, injecting, or inhaling, the vaccine composition of thepresent invention, regardless of form. In some embodiments, a singleadministration of the vaccine composition of the invention is sufficientfor methods as described herein. A single dose of the vaccinecomposition of the invention can result in a sustained immune response,e.g., antibody-mediated and/or cell-mediated immune response, againstthe infected pathogen. In other embodiments, the vaccine compositionsmay be administered in multiple administration, for example, in aprime-boost strategy. In some embodiment, the same vaccines given in theearlier priming immunizations are used for subsequent boostimmunizations. In other embodiments, prime-boost can be done withdifferent types of vaccines containing the same antigens. In someembodiments, a vaccine composition of the invention is administered attime zero and a second vaccine composition of the invention isadministered following a period of time, for example from 10 to 30 days,from 10 to 60 days, or from 10 to 100 days.

Currently developed vaccines usually require multiple immunizations forvaccines to be successful. For a pediatric population, up to fiveimmunizations may be needed, as is the case for Diphtheria, Tetanus andPertussis (DTP) vaccine, which is given three times during the first sixmonths after birth, followed by a fourth dose in the second year oflife, and a final boost between four and six years of age. Still, someof the vaccines need additional boosts even in adults who have alreadyreceived the complete immunization series, for example, theTetanus-diphtheria (Td) vaccine, for which a boost is recommended every10 years throughout a person's lifespan. It is well accepted thatmultiple immunizations (i.e. “prime-boost”) are critical for even themost successful vaccines. This principle applies to live attenuatedvaccines (e.g., oral polio vaccine), inactivated vaccines (e.g.,hepatitis A vaccine), recombinant protein subunit vaccines (e.g.,hepatitis B vaccine) and polysaccharide vaccines (e.g., HaemophilusInfluenzae type b vaccine). However, the vaccine compositions of thepresent invention possess a distinct advantage over existing vaccines inthat only one single administration is sufficient for the methodsdescribed herein. Indeed, as shown in Example 1, a single dose of thevaccine composition of the invention successfully confers a long-termimmune response and protect the vaccinated mice from bacteria challengeover a period of 90 days.

In some embodiments, one or a plurality of vaccine compositions of theinvention is administered to the subject at one or multiple sites.Preferably, each site drains to a lymph node or group of lymph nodes. Insome embodiments, the sites are selected from the group consisting ofthe right arm, the left arm, the right thigh, the left thigh, the rightshoulder, the left shoulder, the right breast, the left breast, theabdomen, the right buttock, and the left buttock. In some embodiments,the site is or drains to a non-encapsulated cluster of lymphoid tissueselected from the group consisting of the tonsils, the adenoids, theappendix, and Peyer's patches. In some embodiments, a vaccinecomposition of the invention is administered to a site that drains tothe spleen.

Any suitable route of administration is encompassed by the methods ofthe invention, e.g. intradermal, subcutaneous, intravenous,intramuscular, or mucosal. Mucosal routes of administration include, butare not limited to, oral, rectal, vaginal, and nasal administration. Insome embodiments, the vaccine composition is administered transdermally,intradermally, subcutaneously, orally, rectally, vaginally or byinhalation. In other embodiments, the vaccine composition is implantedinto a subject.

Where the vaccine composition is in the form of a scaffold, the methodof vaccinating a subject comprises injecting or implanting the scaffoldcomposition in the subject, preferably subcutaneous implantation. Incertain embodiments, the method of vaccinating a subject may compriseimplanting or injecting the scaffold vaccine composition in one or moreareas of the subject's anatomy.

The methods disclosed herein can be applied to a wide range of subjects.In some embodiments, the subject is a mammal, e.g., a human, an embryo,a horse, a dog, a cat, a cow, a sheep, a pig, a fish, an amphibian, areptile, a goat, a bird, a monkey, a mouse, a rabbit, and a rat. Inother embodiments, the subject is a human. In certain embodiments, thesubject is an embryo.

The terms “treat” or “treating,” as used herein, refer to partially orcompletely alleviating, inhibiting, ameliorating, and/or relieving theinfectious disease or condition from which the subject is suffering. Insome instances, treatment can result in the continued absence of theinfectious disease or condition from which the subject is suffering suchas pain or distress.

In some instances, treatments methods can include a singleadministration, multiple administrations, and repeating administrationas required for the prophylaxis or treatment of the infectious diseaseor condition from which the subject is suffering. In some instancestreatment methods can include assessing a level of disease in thesubject prior to treatment, during treatment, and/or after treatment. Insome instances, treatment can continue until a decrease in the level ofdisease in the subject is detected.

The methods herein include administration of an effective amount ofvaccine compositions to achieve the desired or stated effect, e.g.,eliminating infectious pathogens from a subject, thereby treatinginfectious diseases. Specific dosage and treatment regimens for anyparticular patient will depend upon a variety of factors, including theactivity of the specific compound employed, the age, body weight,general health status, sex, diet, time of administration, rate ofexcretion, drug combination, the severity and course of the disease,condition or symptoms, the patient's disposition to the disease,condition or symptoms, and the judgment of the treating physician.

Following administration, the subject can be evaluated to detect,assess, or determine the level of infectious disease. In some instances,treatment can continue until a change, e.g., reduction, in the level ofinfectious disease in the subject is detected.

Upon improvement of a patient's condition, e.g., a change, e.g.,decrease, in the level of disease in the subject, a maintenance dose ofa vaccine composition of the present invention may be administered, ifnecessary. Subsequently, the dosage or frequency of administration, orboth, may be reduced, as a function of the symptoms, to a level at whichthe improved condition is retained. Patients may, however, requireintermittent treatment on a long-term basis upon any recurrence ofdisease symptoms.

In some embodiments, an effective amount of a vaccine composition of theinvention is the amount sufficient to reduce the severity of aninfectious disease or condition in a subject having an infectiousdisease or condition, or the amount sufficient to reduce or amelioratethe severity of one or more symptoms thereof, or the amount sufficientto prevent the progression of the infectious disease or condition, orthe amount sufficient to enhance or improve the therapeutic effect(s) ofanother therapy or therapeutic agent administered concurrently with,before, or after a vaccine composition of the invention.

Symptoms of infectious diseases are well-known to those of skill in theart. Exemplary signs and symptoms of infectious diseases include, butare not limited to, fever, diarrhea, fatigue, muscle aches, coughing,running nose, red and watery eyes, pain, distress, loss of appetite,nausea, abdominal discomfort, weakness, weight loss, and rash.

In some embodiments, the effective amount of a vaccine composition ofthe invention is the amount sufficient to produce an antibody secretingB cell or cytotoxic T cell mediated immune response directed against oneor more of the pathogen or pathogen fragments of the vaccinecompositions of the invention. The ability of the vaccine compositionsof the invention to elicit an immune response can be determined usingany routine method available to those of skill in the art. In someembodiments, the effective amount of each composition is the amountsufficient to produce a cytotoxic T cell response in the subject asmeasured, for example, by a mixed lymphocyte T cell assay.

In some embodiments, the effective amount of the vaccine compositionadministered to the subject, or at a particular site of the subject, isthat amount which delivers 1 picogram to 1000 micrograms of the one ormore pathogens or pathogen fragments of the composition. In someembodiments, the amount of pathogens or pathogen fragments is about 1 pgto about 1000 μg. In some embodiments, the amount of pathogens orpathogen fragments is 1 μg to 100 μg, 1 μg to 200 μg, 1 μg to 300 μg, 1μg to 400 μg, 1 μg to 500 μg, 1 μg to 600 μg, 1 μg to 700 μg, 1 μg to800 μg, or 1 μg to 900 μg. In some embodiments, the amount of pathogensor pathogen fragments is 1 μg to 10 μg, 1 μg to 20 μg, 1 μg to 30 μg, 1μg to 40 μg, 1 μg to 50 μg, 1 μg to 60 μg, 1 μg to 70 μg, 1 μg to 80 μg,or 1 μg to 90 μg. In some embodiments, the amount of pathogens orpathogen fragments is 1 pg to 100 μg, 1 pg to 90 μg, 1 pg to 80 μg, 1 pgto 70 μg, 1 pg to 60 μg, 1 pg to 50 μg, 1 pg to 40 μg, 1 pg to 30 μg, 1pg to 20 μg or 1 pg to 10 μg. In other embodiments, the amount ofpathogens or pathogen fragments is 10 pg to 1 μg, 20 pg to 1 μg, 30 pgto 1 μg, 40 pg to 1 μg, 50 pg to 1 μg, 60 pg to 1 μg, 70 pg to 1 μg, 80pg to 1 μg, 90 pg to 1 μg, 100 pg to 1 μg, 1000 pg to 1 μg.

The present invention also provides methods for treating a pathogeninfection in a subject in need thereof. The methods compriseadministering the scaffold composition and the opsonin-bound orlectin-bound pathogen construct as described herein, thereby treatingthe pathogen infection in the subject.

In another aspect, the present invention provides methods of vaccinatinga subject against a pathogen infection. The methods compriseadministering the scaffold composition and the opsonin-bound orlectin-bound pathogen construct as described herein, thereby vaccinatingthe subject against the pathogen infection.

In one aspect, the present invention provides methods of treating anantibiotic-resistant bacterial infection in a subject in need thereof.The methods comprise administering the scaffold composition and theopsonin-bound or lectin-bound pathogen construct as described herein,thereby treating the antibiotic-resistant bacterial infection in thesubject. In some embodiments, the vaccine composition is specific forthe antibiotic-resistant bacterium in the subject.

In another aspect, the present invention provides methods of decreasingthe level of a pathogen in a subject having a pathogen infection. Themethods comprise administering the scaffold composition and theopsonin-bound or lectin-bound pathogen construct as described herein,thereby decreasing the level of the pathogen in the subject. In someembodiments, the level of the pathogen is decreased in an organ of thesubject. In some embodiments, the organ is selected from the groupconsisting of a lung, a liver, a kidney, and a spleen.

In one aspect, the present invention provides methods of increasing thesurvival rate of a subject having a pathogen infection. The methodscomprise administering the scaffold composition and the opsonin-bound orlectin-bound pathogen construct as described herein, thereby increasingthe survival rate of the subject.

In another aspect, the present invention provides methods of reducingthe level of distress associated with a pathogen infection in a subjectin need thereof. The methods comprise administering the scaffoldcomposition and the opsonin-bound or lectin-bound pathogen construct asdescribed herein, thereby reducing the level of distress associated witha pathogen infection in the subject.

In a further aspect, the present invention provides methods of reducingthe level of pain associated with a pathogen infection in a subject inneed thereof. The methods comprise administering the scaffoldcomposition and the opsonin-bound or lectin-bound pathogen construct asdescribed herein, thereby reducing the level of pain associated with apathogen infection in the subject.

In some embodiments, the scaffold composition and the opsonin-bound orlectin-bound pathogen construct are administered simultaneously to thesubject. In other embodiments, the scaffold composition is administeredto the subject separately from, e.g., prior to or after, theopsonin-bound or lectin-bound pathogen construct.

The present invention also provides methods of producing a vaccine. Themethods comprise contacting a sample comprising a pathogen or fragmentthereof with an opsonin or lectin, wherein the opsonin or lectin iscapable of binding to the pathogen or fragment thereof in the sample,thereby forming an opsonin-bound or lectin-bound pathogen construct;isolating the opsonin-bound or lectin-bound pathogen construct from thesample; and combining the opsonin-bound or lectin-bound pathogenconstruct with a bioagent capable of recruiting an immune cell in asubject, thereby producing the vaccine.

In another aspect, the present invention provides methods of producing avaccine, comprising contacting a sample comprising a pathogen orfragment thereof with an opsonin or lectin, wherein the opsonin orlectin is capable of binding to a pathogen or fragment thereof in thesample, thereby forming an opsonin-bound or lectin-bound pathogenconstruct; isolating the opsonin-bound or lectin-bound pathogenconstruct from the sample; and combining the isolated opsonin-bound orlectin-bound pathogen construct with a scaffold, thereby producing thevaccine.

In yet another aspect, the present invention provides methods ofproducing a vaccine, comprising administering an opsonin or lectin to asubject, wherein the opsonin or lectin is capable of binding to apathogen or fragment thereof, from the subject, thereby forming anopsonin-bound or lectin-bound pathogen construct; isolating theopsonin-bound or lectin-bound pathogen construct from the subject; andcombining the isolated opsonin-bound or lectin-bound pathogen constructwith a scaffold, thereby producing the vaccine.

In some embodiments, the pathogen is derived from a subject in vivo. Inother embodiments, the pathogen is derived from an in vitro culture, amicroorganism lysate, a crude lysate, or a purified lysate. In certainembodiments, the pathogen is a synthetic pathogen.

In some embodiments, the pathogen comprises a pathogen-associatedmolecule pattern (PAMP). In some embodiments, the PAMP is selected fromthe group consisting of a pathogen fragment, a pathogen debris, apathogen nucleic acid, a pathogen lipoprotein, a pathogen surfaceglycoprotein, a pathogen membrane component, and a released componentfrom the pathogen.

V. Kits

The invention also provides a kit for vaccinating a subject against apathogen infection. Such kits can include a composition describedherein. Such kits can also facilitate performance of the methodsdescribed herein.

In one aspect, the kit comprises the vaccine compositions of theinvention and instructions for administering the vaccine compositions toa subject. In some embodiments, the vaccine composition is prepackagedin a sterile container.

In another aspect, the kit comprises a scaffold composition and anopsonin-bound or lectin-bound pathogen construct of the invention, andinstructions for administering the scaffold composition and theopsonin-bound or lectin-bound pathogen construct to the subject. In someembodiments, the scaffold composition or the opsonin-bound orlectin-bound pathogen construct are prepackaged in a sterile container.The scaffold composition or the opsonin-bound or lectin-bound pathogenconstruct can be prepackaged in different sterile containers or in thesame sterile container.

The composition in each container may be in the form of apharmaceutically acceptable solution, e.g., in combination with sterilesaline, dextrose solution, or buffered solution, or otherpharmaceutically acceptable sterile fluid. Alternatively, thecomposition may be lyophilized or desiccated. The kit optionally furthercomprises in a separate container a pharmaceutically acceptable solution(e.g., saline, dextrose solution, etc.), preferably sterile, toreconstitute the composition to form a solution for injection purposes.

The kits of the invention may optionally comprise additional componentsuseful for performing the methods of the invention. The kit may compriseone or more reusable or disposable device(s) for administration (e.g.,syringes, needles, dispensing pens), preferably packaged in sterileform, and/or a packaged alcohol pad.

In certain embodiments, kits can be supplied with instructionalmaterials which describe performance of the methods of the invention.Kits may include instructions for administration or delivery of avaccine composition by a clinician or by the patient. In anotherembodiment, the kits may include instructions for proper storage andhandling of the vaccine compositions.

The present invention is further illustrated by the following examples,which are not intended to be limiting in any way. The entire contents ofall references, patents and published patent applications citedthroughout this application, as well as the Figures, are herebyincorporated herein by reference.

EXAMPLES Example 1 Infection Vaccine Study

Preparation of FcMBL Beads

Magnetic FcMBL beads were used to capture pathogens or pathogenfragments, e.g., pathogen associated molecular patterns (PAMPs).Briefly, 1 μM Streptavidin coated superparamagnetic beads (Dynal, LifeTechnologies) was coupled with FcMBL. FcMBL is an engineered lectinconsisting of the carbohydrate recognition domain (CRD) of mannosebinding lectin (MBL) fused to the Fc region of human IgG with anengineered amino-oxy biotin site. The FcMBL fusion protein was orientedon the beads using the amino-oxy biotin on the terminal of the Fc regionof FcMBL. Other beads have been tested for coupling to FcMBL and usedfor capture of pathogens or pathogen fragments, for example, 500 nmAdemtech superparamagnetic beads. Direct coupling of FcMBL to the beadswithout Streptavidin were also tested and shown to work for pathogencapture. Final concentration of beads used for vaccine generation was 5mg/mL.

Preparation of Pathogen Samples

Pathogen for vaccines can be isolated from any infection. For example,pathogen can be cultured for PAMPs preparation. Alternatively, PAMPs canbe captured directly from blood sample. Experiments in the present studywere performed using a MDR strain of pathogenic RS218 E. coli, isolatedfrom infantile spinal meningitis. RS218 has the serotype O18ac:H7:K1.

RS218 E. coli were grown in RPMI media supplemented with 10% glucose to0.5 McF (1e8 CFU/mL). RPMI media was used to avoid yeast extracts andother components that could interfere with FcMBL capture or compromisevaccine generation. Sufficient bacteria (a minimum of 1 mL of bacteriasolution for each scaffold) were treated with 1 mg/mL Cefipime and 500μg/mL Amikacin for 24 hours. Full killing of bacteria was confirmed byovernight plating where 0 CFU was detected

The cell wall pathogen associated molecular patterns (PAMPs) of RS218were captured using magnetic opsonins coated with FcMBL in combinationwith a biospleen dialysis-like therapeutic device. Captured RS218 werethen included in a therapeutic vaccine using a PLGA scaffold in thepresence or absence of CpG and GM-CSF adjuvants for in vivo mouse modelstudies.

The PAMPs captured by FcMBL were carbohydrate containing membranecomponents or fragments of RS218, for example, blebs, vesicles or LPS.To quantify PAMPs captured by FcMBL, a standard curve was generatedusing the fungal MBL target, mannan (FIG. 2). Briefly, 1 μMsuperparamagnetic beads coated with FcMBL were used to capture mannan ineither buffer or whole donor blood. Serial dilutions of mannan weremixed with the FcMBL beads, and quantified by ELISA. RS218 cell wallfragments were quantified as MBL-bound pathogen associated molecularpatterns (mPAMPs) by interpolation of the standard curve (FIG. 3). ForRS218 bacteria, 15 ng/mL PAMPs on 25 μL/mL beads were chosen forsubsequent vaccine generation.

Preparation of FcMBL-RS218 PAMPs Beads

Based on PAMPs quantification of antibiotics treated RS218 bacteria, 250μl of beads (5 mg/mL) were added to 10 mL of diluted killed bacteriasolution (equivalent to 15 ng/mL PAMPs) and incubated for 20 minutes.Beads were removed magnetically, washed 1× in TBST 5 mM Ca⁺⁺ andresuspended in 10 mL of TBST 5 mM Ca⁺⁺ for vaccine generation. Absenceof live bacteria was reconfirmed by plating (0 CFUs). Samples werestored at −80° C.

The amount of captured RS218 cell wall fragments was quantified asMBL-bound pathogen associated molecular patterns (mPAMPs) using theFcMBL ELISA (FIG. 4). Pre- and post-capture antibiotics treated RS218solution was screened to determine if the amount of mPAMPs in RS218 wasthe same upon treatment with the FcMBL beads. 100 μL of RS218 sampleswere tested. As shown in FIG. 4, RS218 captured by FcMBL beads had asimilar amount of mPAMPs when compared to the no bead control (RS218pre-bead sample). EDTA control was included to demonstrate thecalcium-dependence of mPAMP capture by FcMBL beads suggesting a specificbinding between mPAMPs and FcMBL beads. Endotoxin capture was alsoquantified by comparison of the pre- and post-capture antibioticstreated RS218 solution (Table 1).

TABLE 1 Quantification of Endotoxin Captured by FcMBL Beads EndotoxinUnits (EU/ml) RS218 ABX Pre-Bead 3290 Post-bead Supernatant 1300Preparation of Vaccine Devices Using PLG Scaffolds and FcMBL CapturedRS218

The vaccine devices used in the study were manufactured in accordancewith the protocols developed for a standard PLG melanoma scaffold basedon the WDVAX clinical trial. The FcMBL beads were incorporated into thescaffold similar to the incorporation of melanoma tumor lysate.

The PLG spheres used in this study were 30 μm poly(lactic-co-glycolicacid) (PLGA) spheres. For the control sham device, 180 mg blank PLGspheres were mixed with 1.3 g of sieved sucrose (250-400 μm). Theresultant mixture was weighed and equal amounts were weighed out toenable the manufacture of 10 control sham devices.

For the beads/pathogen device (PLG scaffolds and FcMBL captured RS218),180 mg blank PLG spheres were added to a 10 ml suspension of FcMBLcaptured RS218 cell wall fragments, mixed thoroughly, frozen andlyophilized for approximately 7 days. The lyophilized powder (304 mg)was mixed with 1.3 g of sieved sucrose (250-400 μm). The resultantmixture was weighed and equal amounts were weighed out to enable themanufacture of 10 bead/pathogen devices.

For full vaccine compositions, 180 mg PLG spheres with granulocytemacrophage colony-stimulating factor (GM-CSF) were added to a 10 mlsuspension of FcMBL captured RS218 cell wall fragments, mixedthoroughly, frozen and lyophilized for approximately 7 days. Thelyophilized powder (310 mg) was mixed with 1.3 g sieved sucrose (250-400μm) and 300 mg CpG. The CpG was condensed using polyethylenimine (PEI).The resultant mixture was weighed and equal amounts were weighed out toenable the manufacture of 10 vaccine compositions.

To make the vaccine compositions, the appropriate quantity oflyophilized powders were placed into a 8 mm diameter die and formedunder 1500 psi using a hydraulic press. Once formed, the compositionswere foamed in a pressure chamber by means of exposure overnight to 800psi CO₂ followed by a rapid pressure release. All compositions werestored at −20° C. until ready for animal implantation.

Preparation of Vaccine Devices Using PLG Scaffolds and FcMBL Captured E.cloacae

Preparation of E. cloacae was done using the same method as listed abovefor E. coli RS218. In brief, E. cloacae is grown to 1e8 CFU/mL, treatedwith Cefepime (1 mg/mL) and Amikacin (500 μg/mL), and complete death ofpathogen is confirmed by plating PAMPs are captured using 1 μM FcMBLBeads and quantified by FcMBL ELLecSA (Cartwright et al. EBioMedicine 9(2016) 217-227). Beads containing PAMPs are then incorporated in PLGscaffold.

For manufacture of PLGA vaccine scaffolds, 18 mg PLG microspherescontaining encapsulated GM-CSF (9 μg) were mixed until a homogenouspowder was achieved with a solution containing 15 PAMPS, and then frozenusing liquid N₂ and lyophilized below 100 militorr overnight. Theresulting powder was thoroughly mixed with 30 mg CpG and 130 mg ofsucrose prior to device pressing and high pressure CO₂ foaming. (7-900PSI) The resulting devices were leached in WFI for 3 hours prior toimplantation.

Preparation of Vaccine Devices Using MPS Scaffolds and FcMBL CapturedRS218

Preparation of FcMBL captured RS218 beads is the same as describedabove. For manufacture of MPS vaccine scaffolds. 3 μg of GM-CSF, 10 mgof CpG and a solution containing 15 PAMPS were loaded onto 10 mg of MPSsand mixed vigorously overnight at room temperature using a rotary mixer.The MPSs were then lyophilized and reconstituted in water for injection(WFI) prior to injection.

PLG Scaffold Vaccination Protects Mice from Lethal R5218 Infection

To study the efficacy of the vaccine compositions, mice were firstimmunized with the vaccine compositions, and then challenged with asub-lethal dose of RS218 bacteria. Specifically, RS218 captured usingmagnetic FcMBL beads were incorporated into a PLG scaffold with CpG andGM-CSF adjuvants to generate a full vaccine composition (FIG. 5A). FcMBLbeads were clearly visible dispersed throughout the holes and cavitiesin the PLG scaffold (FIG. 5B). The vaccine compositions were thenimplanted into mice subcutaneously. Sham devices containing the scaffoldalone were used as a negative control. Beads/pathogen devices whereRS218 captured by FcMBL beads were incorporated into the PLG scaffoldwithout any adjuvants were also included in the assay. 10 mice wereincluded in each treatment group.

21 days after immunization, mice were challenged with a high, butsub-lethal dose of RS218 (5e6 CFU per mouse) by intraperitonealinjection for 48 hours. Mice were humanely sacrificed at 12 hours postinfection or earlier if clinical conditions required. Humane endpointoccurred when clinical score dropped to ⅖ and when animals did notrecover or experienced severe pain and distress unalleviated by nursingcare.

Survival Curve Assay

Survival rate was monitored in RS218 infected mice. As demonstrated inFIG. 6, mice treated with full vaccine compositions (PLG scaffolds withFcMBL beads captured RS218 and GM-CSF/CpG) and beads/pathogen devices(PLG scaffold with FcMBL bead captured RS218 without GM-CSF/CpG)experienced a longer survival time than mice treated with sham devices(scaffold alone). 9 out of 10 mice survived after 48 hours when theywere immunized with the full vaccine compositions. In comparison, 50% ofmice receiving sham devices died about 10 hours after injection with thesub-lethal dose of RS218 bacteria, and half of the mice immunized withthe beads/pathogen devices died after 20 hours. These results suggestedthat addition of adjuvants such as GM-CSF and CpG enhanced the efficacyof the vaccine compositions resulting in an increased survival time, andboth the FcMBL captured pathogen and the adjuvants were needed forprotective vaccination.

Organ Pathogen Counts

To quantify the pathogen load within selected organs in RS218 infectedmice, organ cultures were collected in a sterile fashion, processed bymechanical disruption and plated. Briefly, organs harvested in a sterilefashion were added to bead mill tubes. 1 mL of sterile saline was addedto each tube containing the beads and the organ. Each tube was weightedand each tube was bead milled for 5 minutes at a frequency of 30 persecond. Liquefied organs were tittered 1:100 and 1:10,000. Undiluted,1:100 and 1:10,000 organ samples were spiral plated on sheep blood agar,grown overnight at 37° C. and the pathogen load was determined. Theorgan pathogen counts in infected mice were described in Table 2. FIG.7A demonstrates that the overall titer of pathogen was significantlyreduced by 2.5-3.5 logs in mice receiving the full vaccine compositions(p=0.0021-0.0057), whereas no statistically significant difference wasobserved for mice receiving the beads/pathogen device and the shamcontrol. Similar results were observed for pathogen load in individualorgans. As demonstrated in FIG. 7B, the levels of RS218 in lung, liver,kidney and spleen were all significantly lower in mice receiving thefull vaccine compositions (PLG scaffolds with FcMBL beads captured RS218and GM-CSF/CpG), while mice receiving the beads/pathogen devices had asimilar level of pathogens as the control mice, further suggesting thataddition of adjuvants such as GM-CSF and CpG enhanced the efficacy ofthe vaccine compositions and were necessary for protective vaccination.

TABLE 2 Organ Pathogen Counts in Infected Mice Time Scaffold Lung LiverKidney Spleen (hours) Mouse 4-1 1.32E+07 8.93E+08 3.11E+08 4.68E+08 8Mouse 4-4 1.00E+07 1.82E+08 3.54E+07 4.47E+08 8 Mouse 6-3 3.07E+073.63E+07 1.68E+07 3.86E+07 11 Mouse 2-2 3.86E+07 1.19E+09 1.53E+097.87E+08 24 Mouse 2-5 2.61E+09 1.26E+09 6.24E+08 1.45E+09 24 Mouse 3-31.59E+09 1.76E+09 1.24E+09 3.11E+09 12 Mouse 1-4 2.27E+06 3.16E+081.86E+07 2.71E+07 22 Mouse 1-1 3.27E+03 8.24E+08 8.27E+03 8.24E+03 48Mouse 5-2 2.23E+07 1.35E+08 3.96E+08 4.16E+07 36 Mouse 5-5 7.25E+075.33E+04 2.38E+05 9.77E+04 48 Scaffold & FcMBL/RS218 Mouse 4-2 2.36E+077.07E+08 9.52E+08 2.66E+08 9 Mouse 3-1 2.83E+06 2.69E+07 1.35E+085.11E+08 11 Mouse 5-3 2.41E+07 5.36E+08 1.36E+09 8.28E+08 11 Mouse 6-43.18E+08 3.99E+06 3.59E+06 9.89E+06 11 Mouse 2-3 2.22E+07 2.95E+082.58E+07 3.11E+07 22 Mouse 3-4 6.04E+08 1.43E+09 4.55E+08 1.59E+09 22Mouse 1-5 8.22E+06 1.38E+07 3.34E+07 3.95E+07 11 Mouse 1-2 1.72E+097.34E+08 1.19E+08 1.21E+09 12 Mouse 4-5 8.40E+02 1.96E+04 1.88E+031.65E+03 48 Scaffold & FcMBL/RS218 & GM-CpG Mouse 6-5 2.30E+06 2.24E+071.87E+07 9.59E+07 11 Mouse 1-3 7.18E+04 3.57E+08 7.75E+03 3.97E+03 48Mouse 2-1 5.83E+06 5.43E+04 3.86E+03 7.08E+03 48 Mouse 2-4 3.47E+049.00E+04 4.17E+03 5.91E+03 48 Mouse 3-2 2.77E+04 1.62E+04 5.75E+034.02E+03 48 Mouse 3-5 1.82E+03 5.92E+03 9.37E+03 2.10E+03 48 Mouse 4-35.41E+02 7.98E+02 9.22E+02 2.62E+03 48 Mouse 5-1 8.23E+05 1.80E+047.32E+03 4.89E+04 48 Mouse 5-4 3.63E+04 1.59E+05 1.07E+04 6.56E+04 48Mouse 6-2 1.07E+03 1.06E+03 5.24E+02 1.57E+03 48Comparison Between Vaccine with FcMBL Captured RS218 and Vaccine withRS218 Lysate

To determine whether the observed protective effect of the full vaccinecompositions can be reproduced with a lysate of RS218 without the FcMBLbeads, PLG vaccine scaffolds with CpG/GM-CSF containing either FcMBLcaptured RS218 fragments or whole RS218 lysate alone were prepared.

Mice were divided into three treatment groups and PLG scaffolds wereimplanted subcutaneously for 21 days. Group 1 (n=10) received the shamdevice (scaffold and CpG/GM-CSF), Group 2 (n=10) received the scaffoldswith CpG/GM-CSF containing FcMBL beads with captured RS218 fragments,and Group 3 (n=10) received the scaffolds with CpG/GM-CSF containing thewhole RS218 lysate. Same amount of RS218 was used in Groups 2 and 3.

21 days after immunization, mice were challenged with a lethal dose ofRS218 (with a 90% lethal dose (LD90) at 20 hours) by intraperitonealinjection and followed for 48 hours. As shown in FIG. 8, mice receivingboth Group 2 and Group 3 vaccine compositions were protected againstRS218 infection and had a prolonged survival time when compared to micereceiving the sham controls. 9 out of 10 mice receiving the Group 2vaccine compositions and 10 out of 10 mice receiving the Group 3 vaccinecompositions survived, whereas 9 out of 10 mice receiving the shamcontrols were euthanized at about 12 hours to avoid excessive pain andsuffering.

Results from organ pathogen counts further suggested that vaccinecompositions comprising either FcMBL beads with captured RS218 fragments(Group 2) or the whole RS218 lysate (Group 3) were effective inprotecting mice from RS218 infection. FIG. 9 demonstrated that micereceiving both Groups 2 and 3 vaccine compositions had significantlyless pathogen in the organs than mice in the sham vaccinated Group 1.For Group 2 mice, CFU/g titers dropped by 4-6 logs in lung, kidney,spleen, and by about 2 logs in liver. For Group 3 mice, CFU/g titersdropped by 3-4 logs in lung, liver, kidney and spleen. Accordingly, thelevels of RS218 in the majority of organs, i.e., lung, kidney andspleen, were further reduced in vaccinated mice from Group 2 whencompared to mice in Group 3, with the single exception of liver.

These results indicate that both the bacterial lysate and FcMBL beadcaptured bacterial fragments may be used in combination with PLGscaffolds to generate functional vaccine compositions and protect micefrom a lethal challenge of infection. However, vaccine compositions withFcMBL bead captured pathogen fragments exhibited a significant advantageover the direct use of bacterial lysate because no side effect such asleaching lysate and formation of abscess was observed.

As shown in FIG. 10C, a significant endotoxin leakage was observed inmice implanted with the PLG vaccine scaffolds containing the whole RS218lysate (Group 3), whereas the PLG vaccine scaffolds containing the FcMBLcaptured RS218 fragments demonstrated no such leakage (Group 2). Inaddition, at 48 hour sacrifice, formation of large abscesses wasobserved surrounding the scaffolds in Group 3 mice and the scaffoldcould not be separated from adhesions (FIG. 11C). In contrast, thevaccine compositions in Group 2 mice were largely intact and clean, withlittle signs of eroding (FIG. 11B) and Group 1 control mice had intactscaffold with no signs of immune reaction (FIG. 11A). 28 days aftervaccination, the sham vaccine (Group 1) and vaccine compositionscomprising FcMBL beads with captured RS218 fragments (Group 2) werestill present, however, where the RS218 lysate was incorporated directlyinto the PLG scaffold (Group 3), there was a strong local inflammatoryreaction and the scaffold was degraded away, and these mice hadabscesses at the vaccine sites.

The levels of CpG and GM-CSF leakage from the PLG vaccine scaffolds werealso quantified and compared between the three treatment groups. FIG.10A and FIG. 10B demonstrated that no significant difference wasobserved for CpG and GM-CSF leakage.

Therefore, although vaccines compositions comprising both the bacteriallysate and FcMBL bead captured bacterial fragments were shown to protectmice from a lethal challenge of bacteria infection, the vaccinecompositions with FcMBL bead captured pathogen fragments presented asafer, more controlled and localized option without compromising theefficacy of the vaccine compositions.

Cross-Reactivity of Vaccine Compositions with FcMBL Captured Pathogens

To determine whether the vaccine compositions of the invention can beused against different species of a pathogen, PLG vaccine scaffoldscontaining CpG/GM-CSF and FcMBL captured E. cloacae lysates wereprepared using the same method as listed above. In brief, E. cloacae isgrown to 1e8 CFU/mL, treated with Cefepime (1 mg/mL) and Amikacin (500μg/mL), and complete death of pathogen is confirmed by plating. PAMPsare captured using FcMBL Beads and quantified by FcMBL ELLecSA. Beadscontaining PAMPs are then incorporated in PLG scaffold.

Mice were challenged with a lethal dose of RS218 (with a LD90 at 20hours) by intraperitoneal injection. Both E. cloacae and E. coli aremembers of the order Enterobacteriaceae. As shown in FIG. 12A, aprophylactic vaccine of PLG-GMCSF/CpG with FcMBL beads coated with E.cloacae lysate protected 78% mice till the end of the study at 96 hoursfrom the RS218 challenge, while PLG scaffold with only GM-CSF recruitingand CpG adjuvant (without FcMBL beads and bacteria lysate) protectedonly 20% of animals at 96 hours. The vaccine of PLG scaffold containingGM-CSF/CpG and FcMBL beads coated with RS218 lysate protected 100% micetill the end of the study at 96 hours from the RS218 challenge.

In addition, FIG. 12B showed that pathogen counts were reduced in micereceiving the vaccine of PLG-GMCSF/CpG with FcMBL beads coated with E.cloacae lysate.

There results demonstrate that the present vaccine compositions arecapable of targeting against different species or strains of a givenpathogen, thus conferring a significant advantage over existingvaccines.

Long-Term Effect of a Single Implanted Dose of Vaccine Compositions

To determine whether the vaccine compositions of the invention canconfer a long-term protective effect against a pathogen, mice were firstimmunized with a PLG-GMCSF/CpG vaccine composition with FcMBL beadscoated with E. coli RS218 lysate on Day 1, challenged with a sub-lethaldose of RS218 bacteria (LD90 at 20 hours) at Day 21, and rechallenged onDays 60 and 90. As shown in FIG. 15A, all mice receiving the vaccinecompositions survived for more than 90 days, where the control mice diedimmediately after the RS218 challenge at Day 21. These resultsdemonstrate that the vaccine compositions are capable of producing along-term protective effect against the infected pathogen, and a singledose of vaccine compositions can protect the infected subjects for atleast 90 days.

To confirm the sustained effect of the vaccine compositions, mice werevaccinated on Day 1 and then challenged with a higher RS218 dose with a90% lethal dosage at 8 hours on Day 90. FIG. 15B showed that 75% of micereceiving the PLG vaccine compositions survived for more than 120 daysafter the RS218 challenge, whereas the control mice died immediatelyafter the challenge.

MPS Scaffold Vaccination Protects Mice from Lethal R5218 Infection

Vaccine compostitions comprising mesoporous silica (MPS) scaffolds weregenerated. To study the efficacy of the MPS based vaccine compositions,mice were first immunized with the vaccine compositions, and thenchallenged with a sub-lethal dose of RS218 bacteria (LD100 at 36 hours).Specifically, FcMBL captured RS218 fragments were incorporated into aMPS scaffold with CpG and GM-CSF adjuvants to generate a full vaccinecomposition. Vaccine compositions were then injected into mice.Different doses of the captured pathogen (3 PAMP units and 15 PAMPunits) were coated onto the FcMBL beads and then incorporated into theMPS-GMCSF/CpG scaffold. 15 PAMP units of RS218 were also incorproeatedinto a PLG scaffold as a control.

As shown in FIG. 13, MPS scaffolds containing GM-CSF/CpG and 15 PAMPunits protected 90% of mice from 21-days challenge while MPS scaffoldscontaining 3 PAMP units only protected 70%. All the mice receiving PLGscaffolds containing GM-CSF/CpG and FcMBL beads coated with 15 PAMPsalso survived the challenge.

Another set of MPS based vaccines was prepared with a different pathogendosage (7.5 PAMP units). FIG. 14 shows that the MPS vaccine containingGM-CSF/CpG and 7.5 PAMP units protected 90% mice till the end of thestudy at 96 hours from the RS218 challenge (LD90 at 10 hrs) (n=12).However, only 50% of mice vaccinated with sham vaccine survived thebacteria challenge (n=6).

In conclusion, these results demonstrated that PLG or MPS scaffoldsloaded with FcMBL captured pathogens and with adjuvants such asCpG/GM-CSF could function as an effective vaccine composition andprotect mice from a lethal bacterial infection. Mice immunized withthese vaccine compositions exhibited a significantly prolonged survivaltime and a lower organ pathogen count. The ability to combine the FcMBLbead captured pathogens or pathogen associated molecular patterns withthe vaccine scaffolds, e.g., PLG or MPS scaffolds, allows the creationof high potency pathogen vaccines against all types of pathogens, whichcould be of great use in treatment of infectious diseases. In addition,the use of non-infectious vaccines and the ability to prevent leakage ofthe pathogen from the vaccine composition greatly reduced the severeside effects experienced with the leakage of pathogen toxins, thusresulting in a safer, and more controlled vaccine composition. Furthermore, the present vaccine compositions are capable of targeting againstdifferent species or strains of a given pathogen, and producing along-term protective effect against the infected pathogen. Indeed, asingle dose of the vaccine composition of the invention can protect thevaccinated mice from a bacteria challenge over a period of 90 days.

The results were clinically significant as the vaccine compositions andmethods demonstrated the ability to target a specific pathogen in vivofor the generation of immunity, resulting in distinct and protectiveimmune responses.

Example 2 Mechanistic Analysis of the Vaccine Compositions

Additional experiments were performed to determine the kind of immuneresponse involved for the vaccine compositions (PLG or MPS scaffoldsloaded with FcMBL captured pathogens and with adjuvants such asCpG/GM-CSF). Specifically, assays were performed to determine whetherthe vaccine compositions of the invention mediate a cell-mediatedresponse, or an antibody-mediated response. Changes in the levels ofselected cytokines and the levels of specific IgGs, e.g., IgG1 andIgG2a, were measured in response to RS218 infection in the vaccinatedanimals. A major change in the level of cytokines will be an indicationthat the vaccine compositions primarily act through the cell-mediatedresponse, whereas a significant change in IgG levels will suggest anantibody-mediated response.

Histology studies were performed to determine whether the vaccinecompositions of the invention were capable of eliciting a cell-mediatedimmune response against infected pathogens. Mice were implanted with PLGscaffolds with FcMBL beads captured RS218 and GM-CSF/CpG), or injectedwith MPS scaffolds with FcMBL beads captured RS218 and GM-CSF/CpG). Theimplant sites that contained the sham and complete vaccine wereexplanted, embedded, sectioned and stained with Haematoxylin Eosin toidentify infiltrating immune cells. As shown in FIG. 16, no cell wasaccumulated in the control PLG sham device, whereas dense cellularinfiltration were observed with both PLG and MPS scaffolds, suggestingthat a cell-mediated response was initiated by the vaccine compostions.

To determine whether the vaccine compositons mediated anantibody-mediated immune response against infected pathogen, IgG levelswere measured. The analysis was conducted using a Sera ELISA thatdetects anti-RS218 PAMP IgG and IgM antibodies. The 96 well microplatesare coated with RS218 lysate, blocked, and the plates are washed. Thesamples are diluted between 10³-10⁶, then incubated at room temperature.The plates is aspirated and washed, prior to incubation with with HRPgoat anti mouse IgG secondary antibody conjugate. Finally, a TMBSubstrate is added to create an enzymatic reaction and incubated. Thesamples are then analyzed using a spectrophotomter at a wavelength of450 nm. The results of the serial dilutions can then be calculated todetermine the optical density at 0.5 AU, which is used as a conventionfor establishing antibody titers. FIG. 17 shows that IgG titeres weresignificantly increased in mice receiving the vaccine compositions andthe increase in IgG levels lasted for a period of about 90 day, furthersuggesting that a long-term antibody-mediated immune response waselicited by the vaccine compositions.

Example 3 Vaccination Protects Against Multiple Pathogen Infections

Vaccination Protects Mice from Mycobacterium Tuberculosis Infection

Tuberculosis (TB) is an infectious disease caused by the bacteriumMycobacterium tuberculosis (MTb). Tuberculosis generally affects thelungs, but can also affect other parts of the body. Most TB infectionsdo not have symptoms; in which case it is known as latent tuberculosis.About 10% of latent infections progress to active disease which, if leftuntreated, kill about half of those infected. FcMBL can bind tomannosylated components of Mycobacterium tuberculosis (MTb) cell wall,for example, mannose-capped Lipoarabinomannan (ManLAM) andPhosphatidylinositol Mannoside (PIM) (FIG. 18).

To determine whether the vaccine compositions of the present inventionmay be used to protect against tuberculosis, mice were immunized with asingle dose of MPS based vaccine compositions containing GM-CSF/CpG andFcMBL beads coated with mannose-capped Lipoarabinomannan (ManLAM)lysate. FIG. 19 shows that a single dose of the vaccine compositonincreased the titers of LAM-specific IgG by about 2-3 logs overpre-vaccinated naïve animals (p=<0.001). In addition, a significantincrease in the amount of macrophages, CD4+ T cells and dentritic cellswere also observed within the MPS scaffolds (FIG. 20), demonstratingthat a cell-mediated anti-LAM immune response was also produced invaccinated mice. To prepare cells for FACS analysis, single-cellsuspensions from infection vaccine scaffolds were prepared by digestingin collagenase IV (1 mg/ml), for 20 min at 37° C. followed by filteringthrough 70 μm mesh. Spleenic controls for cell gating were mashedthrough a 70 μm mesh and red blood cells were lysed with ACK buffer (150mM NH₄Cl, 1 mM KHCO₃, Na₂EDTA 0.1 mM). Cells were treated with Fc block(anti-mouse CD16/32) and stained with antibodies to CD3e (PE, BDBiosciences), CD4 (PECY7, BD Biosciences), CD8 (FITC, BD Biosciences),The flow cytometry data was analyzed using FlowJo (Tree Star). Thenumbers of CD4⁺ and CD8⁺ T cells in the scaffolds were determined byusing a hemacytometer in combination with cell densities from flowanalyses.

These data demonstrate that the vaccine compostions of the invention arecapable of targeting Mycobacterium tuberculosis and, thus, have a greatpotential in treating tuberculosis.

Vaccination Protects Against Other Pathogens

Since FcMBL is capable of capturing multiple pathogen genera, vaccinecompositions for additional pathogens, for example, gram negativebacteria (E. coli RS218), Gram positive (MRSA) LAM (Mycobacteriumtuberculosis-cell wall component), fungi (Candida albicans), viruses(HIV gp120 antigen) and parasites (Trichomonas vaginalis antigen), weregenerated.

Each viable pathogen is treated with an appropriate antibiotic. MRSA iskilled using Cefepime (1 mg/mL) and Vancomycin (500 μg/mL) and Candidaalbicans is killed using Amphotericin B (1 mg/mL). Preparation of FcMBLcaptured pathogens/antigens coated beads is the same as described abovein sections: Preparation of Pathogen and FcMBL-RS218 PAMPs Beads.

FIG. 21A shows that FcMBL beads were coated with different amount ofpathogen fragments for vaccine preparation. Mice were then vaccinatedwith a single dose of MPS based vaccine compositions containingGM-CSF/CpG and FcMBL beads coated with with samples from the infectiousmicroorganisms. FIG. 21B shows that the titers of LAM-specific IgG wasincreased over pre-vaccinated naïve animals in all cases, demonstratingthat the vaccine compositions were suitable for targeting and mediatingimmune responses against multiple pathogen species.

In addition to antibody-mediated immune response, the vaccinecomposition were able to elicit cell-medisted immune response againsttargeted pathogens. FACS anaklysis of infiltrating cells in splens ofvaccinated animals demonstrated the cell-mediated anti-Trichomonasresponse against Trichomonas lysate incoporated into a vaccine (FIG.22). Control spleens (Naive) showed fewer infiltrating CD4+T cells &CD11c cells than in the vaccinated animal groups.

Example 4 MPS Scaffold Vaccination with FcSPD Captured RS218

An alternative lectin was used to capture pathogen. Pulmonarysurfactants, a complex mixture of lipids and proteins, are essential forlung function. Surfactant protein D (SPD) is another collectin (C-typelectin with collagen region) related to MBL. SPD has a primary role inhost defense of the lung. An FcSPD fusion protein was generated, whichhas 77% protein sequence identity to FcMBL. Preparation of FcSPDcaptured RS218 beads are similar as described above.

As shown in FIG. 23, FcSPD was able to bind the RS218 E. coli and theFcSPD captured RS218, when incorcoporate in the MPS scaffold, alsomounted an immune reaction in vaccinated mice. An increased antibodytiter was observed in vaccinated mice, demonstrating that vaccinecompositions comprising FcSPD-captured pathogens were also capable ofmediating immune response against infected pathogens.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. such equivalents areintended to be encompassed by the following claims. The contents of allreferences, patents and published patent applications cited throughoutthis application are incorporated herein by reference.

We claim:
 1. A vaccine composition, comprising a scaffold comprising abiomaterial; a lectin-bound pathogen construct encapsulated by thescaffold, wherein the lectin-bound pathogen construct comprises a solidsubstrate coupled to an immunoglobulin (IgG) Fc region fused to alectin, or portion thereof; and a pathogen, or portion thereof, bound tothe lectin, or portion thereof; a bioagent which recruits an immune cellin a subject; and an adjuvant which stimulates an immune response to thepathogen, or portion thereof, in the subject.
 2. The vaccine compositionof claim 1, wherein the biomaterial is selected from the groupconsisting of glycosaminoglycan, silk, fibrin, the gelatinous proteinmixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells,poly-ethyleneglycol (PEG), polyhydroxy ethyl methacrylate, polyvinylalcohol, polyacrylamide, poly (N-vinyl pyrolidone), poly(lactic acid),poly glycolic acid (PGA), poly lactic-co-glycolic acid (PLGA), polye-carpolactone (PCL), polyethylene oxide, poly propylene fumarate (PPF),poly acrylic acid (PAA), polyhydroxybutyric acid, hydrolysedpolyacrylonitrile, polymethacrylic acid, polyethylene amine, esters ofalginic acid; pectinic acid; and alginate, fully or partially oxidizedalginate, hyaluronic acid, carboxy methyl cellulose, heparin, heparinsulfate, chitosan, carboxymethyl chitosan, chitin, pullulan, gellan,xanthan, collagen, gelatin, carboxymethyl starch, carboxymethyl dextran,chondroitin sulfate, cationic guar, cationic starch, and combinationsthereof.
 3. The vaccine composition of claim 1, wherein the biomaterialis selected from the group consisting of poly(L-lactide-co-glycolide)acid (PLGA), mesoporous silica, cryogel, and combinations thereof. 4.The vaccine composition of claim 1, wherein the bioagent is selectedfrom the group consisting of interleukin (IL)-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, tumor necrosisfactor (TNF)-alpha, interferon (IFN)-gamma, IFN-alpha, granulocytemacrophage colony stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), Fms-related tyrosine kinase ligand (FTL)-3ligand, CCL19, CCL21, M-SCF, MIF, CD40L, CD3, ICAM, transforming growthfactor (TGF)-beta, cytosine-guanosine oligonucleotide (CpG-ODN),lipopolysaccharides (LPS), Fas ligand, Trail, lymphotactin, Mannan(M-FP), APG-2, Hsp70 and Hsp90.
 5. The vaccine composition of claim 4,wherein the bioagent comprises granulocyte macrophage colony stimulatingfactor (GM-CSF).
 6. The vaccine composition of claim 1, wherein theadjuvant is selected from the group consisting of cytosine-guanosineoligonucleotide (CpG-ODN) sequence, granulocyte macrophage colonystimulating factor (GM-CSF), ovalbumin (OVA), monophosphoryl lipid A(MPL), poly(I:C), MF59, alum, aluminum hydroxide, aluminum phosphate,calcium phosphate hydroxide, Quil A, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), FIA, montanide, adjuvant 65,lipovant, poly (DL-lactide-coglycolide) microspheres, paraffin oil,squalene, virosome, AS03, ASO4, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, STING, Toll-like receptorligand, CD40L, pathogen-associated molecular patterns (PAMPs),damage-associated molecular pattern molecules (DAMPs), Freund's completeadjuvant, Freund's incomplete adjuvant, transforming growth factor(TGF)-beta antibody or antagonists, A2aR antagonists,lipopolysaccharides (LPS), Fas ligand, Trail, lymphotactin, Mannan(M-FP), APG-2, Hsp70 and Hsp90.
 7. The vaccine composition of claim 6,wherein the adjuvant is granulocyte macrophage colony stimulating factor(GM-CSF).
 8. The vaccine composition of claim 6, wherein the adjuvantcomprises a CpG-ODN sequence.
 9. The vaccine composition of claim 8,wherein the CpG-ODN sequence comprises a polyehylenimine (PEI)-CpG-ODNsequence.
 10. The vaccine composition of claim 1, wherein the lectin, orportion thereof, is an engineered lectin, or portion thereof.
 11. Thevaccine composition of claim 1, wherein the lectin, or portion thereof,is selected from the group consisting of a collectin, a ficollin, and amannose-binding lectin (MBL).
 12. The vaccine composition of claim 11,wherein the lectin or portion thereof, comprises amino acid residues 81to 228 of human MBL; or amino acid resides 111 to 228 of human MBL. 13.The vaccine composition of claim 1, wherein the lectin comprisesSurfactant Protein D (SPD).
 14. The vaccine composition of claim 1,wherein the solid substrate is selected from the group consisting of amagnetic bead, a microporous membrane, a hollow-fiber reactor, a bloodfiltration membrane and a blood flow device.
 15. The vaccine compositionof claim 14, wherein the solid substrate is a magnetic bead.
 16. Thevaccine composition of claim 1, wherein the pathogen, or portionthereof, is present in the vaccine composition at a quantity of about 1pg to about 1000 μg.
 17. The vaccine composition of claim 1, wherein thepathogen, or portion thereof, is an infectious microorganism selectedfrom the group consisting of a bacterium, a fungus, a virus and aparasite, or a fragment thereof.
 18. The vaccine composition of claim17, wherein the bacterium is selected from the group consisting ofAcinetobacter baumanii, Burkholderia cepacia, Bacterioides fragilis,Chlamydia trachomatis, Citrobacter freundii, Campylobacter jejuni,Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae,Haemophilus inf b, Helicobacter pylori, Klebsiella oxytoca, K. pneumonia(MDR/CRE), Legionella pneumophila, Neisseria meningitides, Neisseriagonorrhoeae, Pseudomonas aeruginosa, Salmonella typhi, paratyphi,typhimurium, Serratia marcescens, Shigella flexneri, Stenotrophomonasmaltophilia, Yersinia pseudotuberculosis, Bacillus subtilis, Clostridiumneoformans, C. difficile, C. perfringens, Corynebacterium spp,Enterococcus faecalis, Enterococcus faecium, vancomycin-resistantEnterococci (VRE), Listeria monocytogenes, Mycobactrium avium, M.tuberculosis, M. leprae, Nocardia farcinica, P. acnes, Staphylococcusaureus, methicillin-susceptible Staphylococcus aureus (MSSA),methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcusepidermidis, Streptococcus pyogenes, Strep Group A, Strep Group B(agalactiae) and Strep Group C.
 19. The vaccine composition of claim 17,wherein the bacterium is an antibiotic-resistant bacterium or amulti-drug resistant bacterium.
 20. The vaccine composition of claim 19,wherein the antibiotic-resistant bacterium or the multi-drug resistantbacterium is selected from the group consisting of Acinetobacterbaumanii, Escherichia coli, Klebsiella oxytoca, K. pneumonia (MDR/CRE),Pseudomonas aeruginosa, C. difficile, vancomycin-resistant Enterococci(VRE) and methicillin-resistant Staphylococcus aureus (MRSA).
 21. Thevaccine composition of claim 17, wherein the fungus is selected from thegroup consisting of Aspergillus spp, Blastomyces, Candida albicans,glabrata, guilliermondii, krusei, parapsilosis, tropicalis,Cryptococcus, Fusarium spp., Mucor spp., Saccharomyces, and Pneumocystisjirovecii (carinii).
 22. The vaccine composition of claim 17, whereinthe virus is selected from the group consisting of Dengue virus, Ebolavirus, EBV, Hepitis A virus, Hepitis B virus, Hepitis C virus, Hepitis Dvirus, HSV 1, HSV 2, HIV, Cytomegalovirus (CMV), Influenza A virus,Marburg virus, Human respiratory syncytial virus (RSV), SARS-CoV, WestNile virus, Human papillomavirus (HPV), Human rhinoviruses (HRVs), andZica virus.
 23. The vaccine composition of claim 17, wherein theparasite is selected from the group consisting of Cryptosporidium,Leishmania, Malaria, Schistosoma, Trichomonas and Trypanosoma.
 24. Thevaccine composition of claim 17, wherein the pathogen, or portionthereof, comprises a cell membrane component of the infectiousmicroorganism.
 25. The vaccine composition of claim 17, wherein thepathogen, or portion thereof, comprises a whole cell isolated from theinfectious microorganism.
 26. The vaccine composition of claim 1,wherein the pathogen, or portion thereof, is a mycoplasma.
 27. Thevaccine composition of claim 26, wherein the mycoplasma is selected fromthe group consisting of M. pneumoniae, M. hominis and M. orale.
 28. Thevaccine composition of claim 1, wherein the pathogen, or portionthereof, comprises a pathogen-associated molecule pattern (PAMP). 29.The vaccine composition of claim 28, wherein the PAMP is selected fromthe group consisting of a pathogen fragment, a pathogen debris, apathogen nucleic acid, a pathogen lipoprotein, a pathogen surfaceglycoprotein, a pathogen membrane component, and a component releasedfrom the pathogen.
 30. The vaccine composition of claim 29, wherein thecomponent released from the pathogen, or portion thereof, comprises atoxin.
 31. The vaccine composition of claim 30, wherein the toxin isselected from the group consisting of endotoxin, lipopolysaccharide(LPS), lipoteichoic acid (LTA), wall teichoic acid (WTA) and Ricin. 32.The vaccine composition of claim 1, wherein the pathogen, or portionthereof, is in a sample derived from a subject.
 33. The vaccinecomposition of claim 32, wherein the sample is selected from the groupconsisting of a blood sample, a plasma sample, a serum sample, a bloodculture sample, a cerebrospinal fluid sample, a joint fluid sample, aurine sample, a semen sample, a saliva sample, a sputum sample, abronchial fluid sample, and a tear sample.
 34. The vaccine compositionof claim 1, wherein the pathogen, or portion thereof, is derived from anin vitro culture, a microorganism lysate, a crude lysate, or a purifiedlysate.
 35. The vaccine composition of claim 1, wherein the pathogen, orportion thereof, is a synthetic pathogen.
 36. The vaccine composition ofclaim 1, wherein the pathogen, or portion thereof, is neutralized. 37.The vaccine composition of claim 36, wherein the pathogen or portionthereof, is neutralized by treatment with antibiotics, ultravioletlight, sonication, microwave, bead mill, x-ray, autoclave, irradiationor mechanical disruption.
 38. The vaccine composition of claim 37,wherein the pathogen, or portion thereof, is non-infectious afterneutralization.
 39. The vaccine composition of claim 1, wherein theimmune cell is an antigen-presenting cell.
 40. The vaccine compositionof claim 39, wherein the immune cell is selected from the groupconsisting of a dendritic cell, a macrophage, a T cell and a B cell. 41.The vaccine composition of claim 1, wherein the vaccine compositioncomprises at least two different types of pathogens, or portionsthereof.
 42. The vaccine composition of claim 1, wherein the vaccinecomposition is capable of targeting against different species of apathogen.
 43. The vaccine composition of claim 1, wherein the vaccinecomposition is suitable for implantation in a subject; is suitable forinjection in a subject; or is suitable for oral administration in asubject.
 44. The vaccine composition of claim 43, wherein the vaccinecomposition is suitable for subcutaneous implantation.
 45. The vaccinecomposition of claim 43, wherein the vaccine composition is in the formof a pill, a tablet, a capsule, a soft gel, a chewable, a powder, anemulsion, or an aqueous solution.
 46. The vaccine composition of claim1, wherein the vaccine composition is lyophilized.
 47. The vaccinecomposition of claim 46, wherein the vaccine composition has a shelflife of about 30 days to about 1 year; or is capable of being stored atroom temperature.
 48. The vaccine composition of claim 1, wherein thevaccine composition is capable of immobilizing the lectin-bound pathogenconstruct and preventing leakage of the lectin-bound pathogen constructfrom the scaffold.
 49. The vaccine composition of claim 1, wherein theimmunoglobulin Fc region is directly coupled to the solid substrate. 50.The vaccine composition of claim 1, wherein the immunoglobulin Fc regionis indirectly coupled to the solid substrate by an amino-oxy biotin. 51.A vaccine composition, comprising a scaffold comprising a biomaterialselected from the group consisting of poly(L-lactide-co-glycolide) acid(PLGA), mesoporous silica, cryogel, and combinations thereof; alectin-bound pathogen construct encapsulated by the scaffold, whereinthe lectin-bound pathogen construct comprises a bead coupled to animmunoglobulin (IgG) Fc region fused to a lectin, or portion thereof;and a pathogen, or portion thereof, bound to the lectin, or portionthereof; a bioagent which recruits an immune cell in a subject; and anadjuvant which stimulates an immune response to the pathogen, or portionthereof, in the subject.
 52. A vaccine composition, comprising ascaffold comprising mesoporous silica; a lectin-bound pathogen constructencapsulated by the scaffold, wherein the lectin-bound pathogenconstruct comprises a magnetic bead coupled to an immunoglobulin (IgG)Fc region fused to a mannose-binding lectin (MBL), or portion thereof;and a pathogen, or portion thereof, bound to the lectin, or portionthereof; a bioagent comprising granulocyte macrophage colony stimulatingfactor (GM-CSF); and an adjuvant comprising granulocyte macrophagecolony stimulating factor (GM-CSF) and/or a CpG-ODN sequence.
 53. Avaccine composition, comprising a scaffold comprising poly(L-lactide-co-glycolide) acid (PLGA); a lectin-bound pathogen constructencapsulated by the scaffold, wherein the lectin-bound pathogenconstruct comprises a magnetic bead coupled to an immunoglobulin (IgG)Fc region fused to a mannose-binding lectin (MBL), or portion thereof;and a pathogen, or portion thereof, bound to the lectin, or portionthereof; a bioagent comprising granulocyte macrophage colony stimulatingfactor (GM-CSF); and an adjuvant comprising granulocyte macrophagecolony stimulating factor (GM-CSF) and/or a CpG-ODN sequence.
 54. Thevaccine composition of claim 52 or 53, wherein the pathogen or portionthereof, comprises a pathogen-associated molecule pattern (PAMP). 55.The vaccine composition of claim 52 or 53, wherein the pathogen, orportion thereof, is a bacterium.
 56. The vaccine composition of claim55, wherein the bacterium is an antibiotic-resistant bacterium or amulti-drug resistant bacterium.
 57. The vaccine composition of claim 56,wherein the antibiotic-resistant bacterium or the multi-drug resistantbacterium is selected from the group consisting of Acinetobacterbaumanii, Escherichia coli, Klebsiella oxytoca, K. pneumonia (MDR/CRE),Pseudomonas aeruginosa, C. difficile, vancomycin-resistant Enterococci(VRE) and methicillin-resistant Staphylococcus aureus (MRSA).
 58. Thevaccine composition of claim 52 or 53, wherein the vaccine compositionis capable of being stored at room temperature.
 59. A kit forvaccinating a subject against a pathogen infection, comprising a vaccinecomposition of claim 1; and instructions for administering the vaccineto the subject.
 60. A kit for vaccinating a subject against a pathogeninfection, comprising a vaccine composition of claim 52; andinstructions for administering the vaccine to the subject.
 61. The kitof claim 60, wherein the vaccine composition is prepackaged in a sterilecontainer.
 62. A kit for vaccinating a subject against a pathogeninfection, comprising a vaccine composition of claim 53; andinstructions for administering the vaccine to the subject.
 63. The kitof claim 62, wherein the vaccine composition is prepackaged in a sterilecontainer.
 64. A method of treating an antibiotic-resistant bacterialinfection in a subject in need thereof, comprising administering thevaccine composition of claim 1 to the subject, thereby treating theantibiotic-resistant bacterial infection in the subject.
 65. A method ofincreasing the survival rate of a subject having a pathogen infection,comprising administering the vaccine composition of claim 1 to thesubject, thereby increasing the survival rate of the subject.
 66. Amethod of treating a pathogen infection in a subject in need thereof,comprising administering the vaccine composition of claim 1 to thesubject, thereby treating the pathogen infection in the subject.
 67. Amethod of vaccinating a subject against a pathogen infection, comprisingadministering the vaccine composition of claim 1 to the subject, therebyvaccinating the subject against the pathogen infection.
 68. A method ofdecreasing the level of a pathogen in a subject having a pathogeninfection, comprising administering the vaccine composition of claim 1to the subject, thereby decreasing the level of the pathogen in thesubject.
 69. The method of any one of claims 64, 65, 66, 67, and 68,wherein the subject is a mammal.
 70. The method of claim 69, wherein themammal is selected from the group consisting of a human, an embryo, ahorse, a dog, a cat, a cow, a sheep, a pig, a fish, an amphibian, areptile, a goat, a bird, a monkey, a mouse, a rabbit, and a rat.
 71. Themethod of claim 69, wherein the mammal is a human.
 72. The method ofclaim 64, wherein the lectin-bound pathogen construct is specific forthe antibiotic-resistant bacterium in the subject.
 73. The method ofclaim 68, wherein the level of the pathogen is decreased in an organ ofthe subject.
 74. The method of claim 72, wherein the organ is selectedfrom the group consisting of a lung, a liver, a kidney, and a spleen.75. The method of any one of claims 64, 65, 66, 67, and 68, wherein theinfection is an acute infection.
 76. The method of any one of claims 64,65, 66, 67, and 68, wherein the infection is a chronic infection. 77.The method of any one of claims 64, 65, 66, 67, and 68, wherein theinfection causes an epidemic.
 78. The method of any one of claims 64,65, 66, 67, and 68, wherein the infection causes a pandemic.
 79. Themethod of any one of claims 64, 65, 66, 67, and 68, wherein theinfection occurs during a war.